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Salivary and plasmatic oxytocin are not reliable trait markers of the physiology of the oxytocin system in humans

  1. Daniel Martins
  2. Anthony S Gabay
  3. Mitul Mehta
  4. Yannis Paloyelis  Is a corresponding author
  1. Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, United Kingdom
  2. Centre for Human Brain Health, University of Birmingham, United Kingdom
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Cite this article as: eLife 2020;9:e62456 doi: 10.7554/eLife.62456

Abstract

Single measurements of salivary and plasmatic oxytocin are used as indicators of the physiology of the oxytocin system. However, questions remain about whether they are sufficiently stable to provide valid trait markers of the physiology of the oxytocin system, and whether salivary oxytocin can accurately index its plasmatic concentrations. Using radioimmunoassay, we measured baseline plasmatic and/or salivary oxytocin from two independent datasets. We also administered exogenous oxytocin intravenously and intranasally in a triple dummy, within-subject, placebo-controlled design and compared baseline levels and the effects of routes of administration. Our findings question the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid trait markers of the physiology of the oxytocin system in humans. Salivary oxytocin is a weak surrogate for plasmatic oxytocin. The increases in salivary oxytocin observed after intranasal oxytocin most likely reflect unabsorbed peptide and should not be used to predict treatment effects.

Introduction

In the last two decades, a wave of studies have sought to investigate the role of oxytocin in human normal and impaired socio-affective behavior and cognition (Donaldson and Young, 2008; Dyball and Paterson, 1983). Several methodological approaches have been adopted to this effect. Predominantly these include: (i) the measurement of psychological or neurobiological outcomes after the intranasal administration of exogenous oxytocin compared to placebo (Leng and Ludwig, 2016); (ii) genetic association studies between variations in candidate genes of the oxytocin pathway and behavioral and brain phenotypes (Onodera et al., 2015; Verhagen et al., 2020); (iii) the assessment of associations between single measurements of the concentration of endogenous oxytocin in peripheral fluids (mainly blood and saliva) at rest (Valstad et al., 2017) and individual differences in neurobehavioral phenotypes (Crockford et al., 2014), psychiatric disorder status and/or symptom severity (Rutigliano et al., 2016).

The latter approach is based on two assumptions. The first is that single measures of baseline levels of endogenous oxytocin in the biological fluids of peripheral compartments can accurately index oxytocin release in the brain (Valstad et al., 2017). The second is that single measures provide reliable estimates of baseline levels of endogenous oxytocin in plasma or saliva. Definitive answers regarding the first assumption, whether it regards baseline or evoked release following some intervention, remain to be obtained (MacLean et al., 2019).

Here, we investigate the second assumption, which is a prerequisite if single measurements of baseline levels of endogenous oxytocin are to be used as a valid trait markers of the physiology of the human oxytocin system (Wang et al., 2006). Currently, we lack robust evidence that single measures of endogenous oxytocin in saliva and plasma at rest are stable enough to provide a valid trait marker of the activity of the oxytocin system in healthy individuals. Indeed, previous studies have claimed within-individual stability of baseline plasmatic and salivary concentrations of oxytocin in both adults and children based on moderate-to-strong correlations between salivary and plasmatic oxytocin concentrations measured repeatedly within the same individual over time using ELISA in unextracted samples (Feldman et al., 2013; Schneiderman et al., 2012; Gordon et al., 2017). However, these studies have a number of methodological limitations that raise questions about the validity of their main conclusion that baseline plasmatic and salivary concentrations are stable within individuals. First, measuring oxytocin in unextracted samples has been postulated as potentially erroneous, given the high risk of contamination with immunoreactive products other than oxytocin (Szeto et al., 2011). It is conceivable that these non-oxytocin immunoreactive products might constitute highly stable plasma housekeeping proteins (Zhu et al., 2019), which masked the true variability in oxytocin concentrations. Second, a simple correlation analysis cannot provide information about the absolute agreement of two sets of measurements – which would be a more appropriate approach to study within-subject reliability/stability. Third, it is not clear whether these findings generalize beyond the early parenting (Feldman et al., 2013) or early romantic (Schneiderman et al., 2012) periods participants were in when the studies were conducted, since these periods engage the activity of the oxytocin system in particular ways (Gordon et al., 2010). Hence, establishing the validity of salivary and plasmatic oxytocin as trait markers of the activity of the oxytocin system in humans remains as an unmet need. Such evidence is urgently required, given reports that plasma and saliva levels of oxytocin are frequently altered during neuropsychiatric illness and that they co-vary with clinical aspects of disease (Rutigliano et al., 2016).

The measurement of salivary oxytocin has been proposed as a surrogate for blood plasma levels (Valstad et al., 2017). Compared to blood sampling, saliva collection presents several logistical and measurement advantages (i.e. relatively clean matrix) (Gröschl, 2008). However, the validity of this approach remains unclear. While elevations in plasmatic oxytocin after intranasal administration are likely to result from capillary absorption in the nasal cavity (Gossen et al., 2012), elevations in saliva oxytocin could result from mucociliary clearance of intranasally delivered oxytocin from the nasal cavity to the oropharynx (‘drip-down’ oxytocin) (Huffmeijer et al., 2012; van Ijzendoorn et al., 2012). This question can be illuminated by ascertaining changes in salivary oxytocin following the intravenous administration of oxytocin, eliminating the confound of drip-down oxytocin. In a previous study using enzyme-linked immunosorbent assay, the administration of low and medium doses of intranasal oxytocin (8 and 24 IU) elevated oxytocin concentrations in saliva, but the administration of a low dose (1 IU) intravenously had no effect. Moreover, concentrations of oxytocin in plasma and saliva did not correlate at baseline or after the administration of exogenous oxytocin for either route (Quintana et al., 2018). These data suggest that salivary oxytocin is a weak surrogate measure for peripheral blood levels. However, questions remain about whether the same results would have been achieved by using a more sensitive method of quantification, such as radioimmunoassay (currently the gold-standard for oxytocin quantification [McCullough et al., 2013]), or higher doses of oxytocin administered intravenously.

Here, we aimed to characterize the reliability of both salivary and plasmatic single measures of basal oxytocin in two independent datasets, to gain insight about their stability in typical laboratory conditions and their validity as trait markers for the physiology of the oxytocin system in humans. Additionally, we investigated whether salivary oxytocin concentration reflects plasmatic oxytocin by examining (i) if the intravenous administration of a large dose of oxytocin which produces sustained increases in plasmatic oxytocin over the course of 2 hr also increases the concentration of salivary oxytocin; (ii) how potential changes in salivary oxytocin compare between different routes of administration (intranasal versus intravenous) and methods of intranasal administration (spray versus a nebuliser); and (iii) the correlation between plasmatic and salivary oxytocin levels at baseline and after the administration of exogenous oxytocin using two different methods of intranasal administration (spray versus nebuliser) and the intravenous route. For all analyses, we followed current gold-standard practices in the field and assayed oxytocin concentrations using radioimmunoassay in extracted samples, which has shown superior sensitivity and specificity when compared to other quantification methods (McCullough et al., 2013).

Results

Baseline salivary and plasmatic oxytocin concentrations across visits

We did not identify any significant differences in mean concentration of baseline oxytocin in saliva or plasma samples across the four visits of dataset A (Plasma: F(1.47, 22.04) = 0.51, p=0.55; Saliva: F(1.06, 12.82) = 0.88, p=0.38) (Figure 1—figure supplement 1). We also did not observe any differences in mean concentrations of baseline oxytocin in plasma across the two visits of dataset B (T(19) = 0.63, p=0.54). However, in a quick inspection of Figure 1, we can observe that the levels of baseline salivary and plasmatic oxytocin fluctuated considerably from one visit to another in most individuals (Figure 1).

Figure 1 with 2 supplements see all
Within-individual variation of baseline measurements of oxytocin in plasma and saliva samples across visits.

Baseline plasmatic and salivary oxytocin fluctuate from one visit to another for most individuals (Dataset A). We replicated this trend in an independent dataset for plasma (Dataset B). Each colored line represents one individual.

Reliability of single oxytocin measurements in the plasma and saliva

Dataset A

Between-visits reliability analysis across the four visits

We estimated the intra-class correlation coefficient (ICC) of single oxytocin measurements in saliva to be 0.23 and in plasma 0.29. The mean coefficient of variation (CV) was 63% for saliva and 57% for plasma measurements. We estimated the number of measurements that would have been required to achieve good reliability (ICC = 0.80) of a putative averaged measure to be 13.39 for saliva and 9.79 for plasma (Table 1).

Table 1
Estimates of absolute and relative between-visits reliability of oxytocin measurements in saliva and plasma samples.

Absolute and relative reliability were analyzed using the within-subject coefficient of variation (CV) and the intra-class correlation coefficient (ICC), respectively. We also present the number of additional measurements of the same individual that would be theoretically required to achieve different levels of reliability (ICC = X) of a hypothetical averaged measure, based on the initial reliabilities estimated for our datasets A and B. This number was calculated using the Spearman-Brown prediction formula. CI – confidence interval; SD – Standard Deviation; *H0: ICC is not significantly different from 0. N represents the actual size of the sample used to calculate the ICCs and the CVs.

ICCCV mean (SD)Number of samples necessary to achieve an ICC = X of the averaged measurements in a design including multiple samples per individual
N95% CIF test*p-valueX = 0.80 (good reliability)X = 0.70 (moderate)X = 0.50 (fair)
LowerUpper
Study APlasma (N = 16)0.29160.060.59F(15, 45) = 2.610.0157 %9.795.712.45
Saliva (N = 13)0.2313− 0.010.58F(12, 36) = 2.220.3263 %13.397.813.35
Study BPlasma (N = 19)0.49190.060.76F(18, 18) = 2.840.0142 %4.162.431.05
Between-visits reliability analysis for each pair of visits

Detailed descriptions of the ICCs and CV estimated for each pair of the four visits included in our analysis of dataset A are presented in Supplementary file 1 – Table 1. For plasma, we found higher estimates of ICC for the following pairs: visits 1–2: 0.80 and visits 3–4: 0.66 (Supplementary file 1 – Table 1). The CVs were 31% for visits 1–2% and 45% for visits 3–4. The estimated ICC for any of the other pairs of visits was not significantly different from 0 (Supplementary file 1 – Table 1). For saliva, we only found higher estimates of ICC for the pair visits 2–3: 0.82 (Supplementary file 1 – Table 1). The estimated ICC for the remaining pairs was not significantly different from 0 (Supplementary file 1 – Table 1). Please see Figure 1—figure supplement 2 and Supplementary file 1 – Table 1 for correlations between baseline concentrations of oxytocin for each pair of visits of dataset A.

Between-visits reliability analysis controlling for the time-interval between visits

In line with our main analysis, we found poor reliabilities for both salivary and plasmatic oxytocin in a subset of our sample where two consecutive saliva and plasma samples were collected with an exact gap of 7 days. For both plasma and saliva, the estimated ICCs were not significantly different from 0 and the CVs were 40% and 49%, respectively (Supplementary file 1 – Table 2). Variance in the within-subject intervals between samples did not correlate with within-participant variance in oxytocin concentrations across participants neither for plasma (Spearman Rho = 0.406, p=0.118) or saliva (Spearman Rho = −0.524, p=0.065).

Within-visit reliability (placebo visit)

We estimated the within-visit ICC to be excellent 0.92 and the CV to be 20% in the placebo session (Supplementary file 1 – Table 3).

Dataset B

We estimated the ICC to be 0.49 for single measurements of baseline plasmatic oxytocin across the two visits. The mean CV was 42%. The number of measures that would have been required to achieve good reliability of a putative averaged measure was estimated to be 4.16 (Table 1).

Effects of intranasal and intravenous oxytocin administration on salivary and plasmatic oxytocin concentrations

For salivary oxytocin, we found a significant treatment × time interaction (F(3, 36) = 18.29, p<0.001). Post-hoc analyses revealed that the administration of oxytocin either by intranasal spray or nebuliser, but not the administration of intravenous oxytocin or placebo, resulted in significant increases of salivary oxytocin levels from baseline (Baseline vs Post-administration: Spray – t(12) = 7.06, adjusted p<0.001; Nebuliser - t(12) = 7.61, adjusted p<0.001; Intravenous - t(12) = 0.07, adjusted p=0.99; Placebo - t(12) = 0.15, adjusted p=0.99) (Figure 2).

Effects of the administration of intranasal and intravenous oxytocin on salivary (A) and plasmatic (B) oxytocin.

We examined the effects of treatment, time and treatment × time on salivary and plasmatic oxytocin in a two-way analysis of variance. Post-administration samples were collected at 115 min post-dosing. Statistical significance was set to p<0.05. **p=0.001 and ****p<0.001, using Tukey for multiple testing correction during post-hoc investigation of significant interaction effects. Please note that although all the statistical analyses were conducted on log-transformed oxytocin concentrations, here we plot the raw values to facilitate interpretation.

For plasmatic oxytocin, we found a significant time × treatment interaction (F(3, 45) = 3.99, p=0.02). Post-hoc investigations revealed that the intravenous administration of oxytocin resulted in a significant increase in plasmatic oxytocin, but placebo or intranasal administration of oxytocin using either the spray or the nebuliser did not produce any changes from baseline at this time-point (Baseline vs Post-administration: Spray – t(15) = 1.38, adjusted p=0.52; Nebuliser - t(15) = 0.25, adjusted p=0.99; Intravenous - t(15) = 3.73, adjusted p=0.001; Placebo - t(15) = 1.54, adjusted p=0.41) (Figure 2).

Association between salivary and plasmatic oxytocin at baseline and after administration of exogenous oxytocin

We did not find a significant correlation between oxytocin concentrations measured in saliva and plasma at baseline (r = 0.10 – Bootstrap 95% CI [−0.23,0.37], p=0.18, BF = 3.02, N = 63) (Figure 3) or following the administration of exogenous oxytocin (spray: r = −0.21 Bootstrap 95% CI [−0.64,0.32], p=0.43, BF = 2.43, N = 16; nebuliser: r = 0.07 Bootstrap 95% CI [−0.45, 0.57], p=0.65, BF = 3.32, N = 14; intravenous: r = −0.05 Bootstrap 95% CI [−0.52, 0.43], p=0.84, BF = 3.27, N = 17; placebo: r = −0.20 Bootstrap 95% CI [−0.64, 0.32], p=0.45, BF = 2.48, N = 16) (Figure 4). Changes in oxytocin concentrations from baseline to post-administration also did not correlate between saliva and plasma, in any of our treatment conditions (spray: r = 0.134 – Bootstrap 95% CI [−0.25,0.67], p=0.62, BF = 4.68, N = 16; nebuliser: r = 0.10 Bootstrap 95% CI [−0.50, 0.45], p=0.73, BF = 4.71, N = 14; intravenous: r = 0.08 Bootstrap 95% CI [−0.36, 0.68], p=0.76, BF = 5.19, N = 17; placebo: r = −0.16 Bootstrap 95% CI [−0.39, 0.27], p=0.56, BF = 4.47, N = 16).

Association between salivary and plasmatic oxytocin concentrations at baseline.

In this scatter plot, we depict the lack of association between salivary and plasmatic oxytocin concentrations at baseline (before any treatment administration). The density plots on the top of each axis show the distribution of oxytocin concentrations for each treatment level. Please note that although all the statistical analyses were conducted on log-transformed oxytocin concentrations, here we plot the raw values to facilitate interpretation.

Association between salivary and plasmatic oxytocin after administration of intranasal and intravenous exogenous oxytocin.

In these scatter plots, we depict the lack of association between salivary and plasmatic oxytocin concentrations at 120 and 115 min after the administration of intravenous and intranasal oxytocin or placebo, respectively. Each panel depicts data from one out of the four treatment levels. Please note that although all the statistical analyses were conducted on log-transformed oxytocin concentrations, here we plot the raw values to facilitate interpretation.

Discussion

Using radioimmunoassay, a gold-standard method for oxytocin quantification, we showed that i) single measures of baseline oxytocin concentrations in saliva and plasma are not stable within the same individual across different days; (ii) for plasma, we replicated this finding in an independent cohort; (iii) intranasal administration of exogenous oxytocin, despite method of administration, increases salivary oxytocin, but intravenous administration of a considerable dose does not produce any changes; iv) salivary and plasmatic oxytocin do not correlate with each other either at baseline or after the intranasal or intravenous administration of exogenous oxytocin. We discuss our main findings below.

We reported poor reliability indexes for single measurements of baseline oxytocin in both plasma and saliva. This suggests that single measures of baseline oxytocin in either fluid cannot be consistently measured for the same individual across different days. The reliability estimates found in the current study are in the range of those already described for other hypothalamic hormones such as vasopressin (Quintana et al., 2017) or prolactin (Muti et al., 1996). These poor reliabilities are unlikely to be explained by variability in the time-interval between visits of the same individual. Three lines of converging evidence support this conclusion. First, we also found poor reliability indexes for both saliva and plasma when we restricted our analysis to a subset of our sample controlling for the exact number of days spacing visits. Second, we did not find any significant effect of time-interval on our estimated ICCs. If time-interval was driving the poor reliabilities then we would have expected that in our pairwise analyses reliability would be consistently higher for samples closer in time and drop as the time-interval between sessions increases. This was not what we found. Third, variability in the within-subject intervals between samples did not correlate with within-participant variance in oxytocin concentrations across participants.

We note that the mean CV for baseline concentrations of oxytocin in saliva and plasma is higher than four times the intra-assay variability of the radioimmunoassay we used (<10%). Furthermore, in a further analysis assessing the within-session stability of plasmatic oxytocin using two measurements collected 15 min apart from each other in the placebo visit (one sample collected at baseline and the other after the intravenous administration of saline), we found excellent within-session reliability (ICC = 0.92, CV = 20%). Together, this suggests that the low reliability of endogenous oxytocin measurements across visits in the current study results from true intrinsic individual biological variability and not technical variability/error in the method used for oxytocin quantification.

Although we made efforts to minimize variability in the conditions in which biological samples were collected across sessions in both datasets (i.e. asking participants to abstain from exercise the day before study; controlling the time of sample collection; processing samples immediately after collection to minimize differences in peptide degradation between samples), several additional factors might underlie the intra-individual biological variability in plasmatic and salivary oxytocin that we report here. These include differences across participants in sexual activity in the days preceding each visit, perceived stress, the amount of social interactions, or other non-acknowledged biological rhythms conditioning variations in oxytocin secretion throughout time (Jong et al., 2015; Engel et al., 2019). For instance, one study showed that oxytocin is secreted in a pulsatile manner in healthy males even at rest (Baskaran et al., 2017). Therefore, sampling during different phases of the pulsatile release of oxytocin for plasma across sessions could explain the discrepancies in oxytocin measurements observed in the current study across visits. The time-interval between measurements do not seem to significantly impact on the reliability of baseline oxytocin, as suggested by the overlap of the 95% confidence intervals of the ICCs estimated for each pair of sessions. Although higher reliabilities could be identified between some consecutive pairs of sessions (separated by an average of about 6 days from each other: sessions 1–2 and sessions 3–4 for plasma measurements), other consecutive pairs of sessions (for instance, sessions 2–3) did not produce significant reliability estimates. If there are specific reasons explaining the higher reliability indices observed for the specific pairs of sessions, these reasons remain to be elucidated. However, it is not implausible that we might have found higher reliabilities for these specific two pairs by chance, since the 95% confidence intervals for the ICCs for all pairs of samples overlapped.

Our observation of poor reliability questions the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid trait markers of the physiology of the oxytocin system in humans. Instead, we suggest that, at best, these measurements can provide reliable state markers within short time-intervals (5 min in our study). Our data does not support previous claims of high stability of plasmatic and salivary oxytocin within individuals over time. For instance, in one study, Feldman et al., 2013 assessed plasmatic oxytocin in recent mothers and fathers at two time-points spaced 6 months apart during the postpartum period. The authors found strong correlations between the two assessments for both mothers and fathers (Feldman et al., 2013). In another study, Schneiderman et al., 2012 found strong correlations between plasmatic oxytocin concentrations measured at two different instances spaced 6 months apart in both single and individuals recently involved in a new romantic relationship (Schneiderman et al., 2012). Two important differences between these studies and ours are i) the method used for oxytocin quantification and (ii) the particular states participants were in when the studies were conducted. Regarding the first difference, these previous studies used ELISA without extraction, reporting concentrations of plasmatic oxytocin well above the typical physiological range of 1–10 pg/ml detected in extracted samples (in their studies, the authors report concentrations above 200 pg/ml). The inclusion of extraction has been postulated as a critical step for obtaining valid measures of oxytocin in biological fluids (Szeto et al., 2011). Unextracted samples were shown to contain immunoreactive products other than oxytocin (Szeto et al., 2011), which contribute largely to the concentrations of oxytocin estimated by this method. It is possible that these non-oxytocin products might represent highly stable plasma housekeeping molecules (Zhu et al., 2019) that masked the true biological variability in oxytocin concentrations between assessments in these previous studies that we could detect in extracted samples in our study. Regarding the second difference, these previous studies on within-individual stability were conducted during the early parenting (Feldman et al., 2013) or early romantic (Schneiderman et al., 2012) periods, which engage the activity of the oxytocin system in particular ways (Gordon et al., 2010). Instead, we used a normative sample that did not specify these inclusion criteria. Hence, we cannot exclude that during these specific periods the reliability of salivary and plasmatic oxytocin concentrations might be higher. We note though that our sample more closely resembles the samples used the vast majority of studies in the field (which sometimes even exclude participants during early parenthood [Bui et al., 2019). Hence, our estimates of reliability are a better starter point for all studies where specific circumstances potentially affecting the activity of the oxytocin system have not been specified a priori.

Our data poses questions about the interpretation of previous evidence seeking to associate single measurements of baseline oxytocin in saliva and plasma with individual differences in a range of neuro-behavioral or clinical traits. Almost all these previous studies have relied on the collection of single samples from each individual. Our findings suggest measures of oxytocin could be inconsistent and thus it is unlikely that a single sample may accurately represent oxytocin physiology and thus capture relevant inter-individual differences. Reliability of measurements also impacts power to detect correlations against these measurements (Kanyongo et al., 2007). To illustrate this point, in Figure 5 we provide the results of a set of simulations investigating how different reliabilities of oxytocin measurements impact the sample sizes necessary to detect significant correlations between oxytocin concentrations in peripheral fluids and neurobehavioral outcomes of different effect sizes. Given the less-than-perfect reliability of oxytocin measurements (ICC around 0.30) we show here, to detect the small-to-medium (r = 0.20–0.50) correlations typically reported in studies using these measurements (Torres et al., 2018), researchers would need sample sizes between 102 and 651 participants to reach minimally acceptable power (80%). Sample sizes in association studies of endogenous oxytocin measurements are typically below 100 participants (Torres et al., 2018).

Influence of less-than-perfect reliability of oxytocin measurements on the sample sizes required to detect significant endogenous oxytocin-outcome associations in neurobehavioral human oxytocin research.

In this figure, we show that the results of a set of simulations illustrating the impact less-than-perfect reliabilities of endogenous oxytocin measurements in peripheral fluids might have on the sample size required to detect significant oxytocin (A) – outcome (B) correlations of varying effect sizes in research studies. We conduct calculations for a minimally acceptable statistical power of 80% in a two-tailed parametric test. The outcome measure (B) was assumed to present perfect reliability. The figure was generated using the ‘pwr.r.test’ function of the ‘pwr’ R package. We specified ‘r’ according to the attenuation formula below (Nunnally, 1970). ICC -. Intraclass correlation coefficient; r – Pearson’s correlation coefficient.

Turning to our second main finding, salivary and plasmatic oxytocin did not correlate at baseline or after administration of exogenous oxytocin (irrespective of route), replicating previous observations of a null association between measurements in these two compartments (Javor et al., 2014). Furthermore, we could not find any significant correlation between changes in salivary or plasmatic oxytocin from baseline to 115 min after the end of our last treatment administration in any of our four treatment conditions. The lack of significant associations between salivary and plasmatic oxytocin (and respective changes from baseline) was further supported through our Bayesian analyses which demonstrated that given our data the null hypotheses were at least three times more likely than the alternative hypothesis. Two hypotheses could account for the lack of correlation between plasmatic and salivary oxytocin. First, as in all other studies in the field, we did not control or manipulate the rate of saliva flow in the current study. Lipid insoluble molecules, such as oxytocin, enter into saliva mainly via the tight junctions between acinar cells through ultrafiltration (Vining et al., 1983). If oxytocin does reach the saliva compartment through an ultrafiltration mechanism (which depends on saliva flow rate [Gröschl, 2008]) then it is possible that when saliva flow is stimulated, oxytocin measurements may better index its plasmatic concentrations. Second, we cannot discard differences in oxytocin degradation rates between saliva and plasma. Future studies should investigate these hypotheses further.

Studies have been using increases in salivary oxytocin after the intranasal administration of exogenous oxytocin to index systemic absorption and establish putative time-windows during which neurobehavioral effects of oxytocin administration may be expected. If oxytocin increases after intranasal administration reflected systemic absorption and transport from the blood then we would have expected that intravenous oxytocin would have also increased salivary oxytocin. Moreover, we would also have expected that increases in plasmatic and salivary oxytocin after intranasal administration would reflect the typical ratio of their concentration as observed during baseline (with lower concentrations in saliva than in plasma). Our findings were not consistent with these expectations. We could replicate previous evidence that intravenous oxytocin does not increase salivary oxytocin (Quintana et al., 2018) and extended it by showing that the lack of increase in salivary oxytocin is not limited to the specific low dose of intravenous OT that was previously used (1IU) and that it is not driven by the insufficient sensitivity of the OT measurement method which had resulted in more than 50% of the saliva samples being discarded in the previous study (Quintana et al., 2018). Therefore, our data supports the notion that increases in salivary oxytocin after its intranasal administration most likely reflect drip-down oxytocin from the nasal cavity (Quintana et al., 2018). If this is the case then oxytocin elevations in saliva are mainly driven by non-absorbed exogenous oxytocin and therefore their use to estimate levels of systemic absorption or predict treatment effects after intranasal oxytocin administration is not valid. We expect this phenomenon to be particularly pronounced for higher administered volumes. Further studies should examine the impact of different administered volumes on increases in salivary oxytocin.

The lack of increase in salivary oxytocin after the intravenous administration of exogenous oxytocin that was consistently found in our study and in a previous study (Quintana et al., 2018) also raises the question of how oxytocin reaches the saliva if not from the blood. Currently, there is no evidence of direct acinar secretion or direct nerve terminals release of oxytocin to the saliva; therefore, transport from the blood remains as the most plausible mechanism of appearance of oxytocin in the saliva. Clarifying these mechanisms of transport is paramount, given the current hypothesis that salivary oxytocin might be superior to plasma in indexing central levels of oxytocin in the CSF (Martin et al., 2018).

One may argue the absence of significant elevations of oxytocin in saliva after its intravenous administration may be explained by significant differences in the kinetics of oxytocin concentration variation between these two compartments. This may include a significant delay in the elevation of oxytocin in saliva after the beginning of its increase in the plasma (explaining why saliva and plasma concentrations of oxytocin are not correlated after its exogenous administration). While this may be possible, in a previous companion paper we showed that the peak in plasmatic oxytocin occurs immediately after the end of its intravenous administration – 115 min before our post-administration saliva sample collection (Martins et al., 2020). It is therefore unlikely that this large interval of time would not have captured a significant delay in saliva elevation of oxytocin if it really existed, especially when after this time-interval plasma oxytocin still remained elevated, compared to baseline levels.

A strength of our study is the replication of poor reliability for baseline plasmatic oxytocin in a second independent dataset, which strengths our confidence in the robustness of our reliability findings. However, we acknowledge the following limitations. First, we only considered baseline measures in our reliability analyses. As for other hypothalamic-pituitary-adrenocortical markers where evoked measures present higher reliability than baseline measures (Coste et al., 1994), stimuli-evoked release of endogenous oxytocin (i.e. after social interaction, stress, pain) might also present higher reliability. Our conclusions are also restricted to male participants and to the radioimmunoassay method of oxytocin measurement, precluding extrapolations to female populations or to other quantification methods. Also, due to time and logistical constrains during our MRI setup, we could only sample saliva before administration and at the end of the scanning period, leaving our analyses on the effects of the administration of exogenous oxytocin on its concentration in saliva restricted to one single-time point post-administration. It is possible that we may have missed peak increases in saliva oxytocin after the intravenous administration of exogenous oxytocin if they occurred between treatment administration and post-administration sampling. This is unlikely given that the dose we administered intravenously resulted in sustained increases in plasmatic oxytocin over the course of 2 hr. Unless the half-life of oxytocin in saliva is much shorter than in the plasma, it would be surprising to not find any increases in salivary oxytocin after intravenous oxytocin given that concentrations of oxytocin in the plasma were still elevated at the specific time-point of our second saliva sample. Currently, we have no estimate for the half-life of oxytocin in saliva; however, given that previous studies have found evidence of increased salivary oxytocin after single intranasal administrations of 16IU and 24IU oxytocin up to seven hours post-administration (van Ijzendoorn et al., 2012), it is unlikely that the half-life of oxytocin is shorter in the saliva than in the plasma.

In summary, single measurements of baseline levels of endogenous oxytocin in saliva and plasma are not stable in typical laboratory conditions and therefore their validity as trait markers of the physiology of the oxytocin system is questionable. Salivary oxytocin is a weak surrogate for plasmatic oxytocin; hence, salivary and plasmatic oxytocin should not be used interchangeably. Finally, increases in salivary oxytocin after the intranasal administration of exogenous oxytocin most likely represent drip-down transport from the nasal to the oral cavity and not systemic absorption. Therefore, increases in salivary oxytocin after intranasal oxytocin administration should not be used to predict treatment effects.

Materials and methods

Participants

Dataset A included 17 healthy, right-handed, male volunteers (mean age (SD) = 23.75 (5.10); range = 18–34) who contributed samples over four visits, as part of a larger study. Dataset B (independent replication study) included 20 healthy, right-handed, male volunteers (mean age (SD) = 24.8 (3.70); range = 21–37), who contributed samples over two visits as part of a different study (both studies described below). All participants had no history of psychiatric disorders or substance abuse, scored negatively on a screening test for recreational drug use, and did not currently use any medication. In dataset A, we screened participants for psychiatric conditions using the Symptom Checklist-90-Revised (Ruis et al., 2014) and the Beck Depression Inventory-II (Sacco et al., 2016) questionnaires. In dataset B, we used the MINI International Neuropsychiatric Interview (Sheehan et al., 1998). Participants were advised to avoid heavy exercise, alcohol, or smoking the day before scanning and avoid any drink or food within 2 hr before scanning. Both studies were approved by King’s College London Research Ethics Committee (Dataset A: PNM/13/14–163; Dataset B: PNM/14/15–32). For both studies, our sample size and number of samples collected per individual would have allowed us to detect intra-class correlation coefficients (ICC) of at least 0.70 (moderate reliability) with 80% of power (Bujang and Baharum, 2017).

Study design

Dataset A

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Participants were recruited to participate in a double-blind, placebo-control, triple-dummy, cross-over MRI study exploring the effects of various methods of exogenous oxytocin administration on cerebral physiological responses at rest (Martins et al., 2020). Participants received, in counterbalanced order, over four consecutive visits spaced on average 8.80 days apart (SD 5.72; range 3–28), approximately 40IU of intranasal oxytocin, either with a nasal spray or the SINUS nebulizer (PARI GmbH), 10IU of oxytocin intravenously, or placebo. The administration of 10IU of oxytocin intravenously produces sustained increases in the levels of plasmatic oxytocin over a 2 hr course (Martins et al., 2020). This aspect of our design would allow us to discard the possibility that lack of changes in salivary oxytocin are due to under dosing. In each visit, blood samples were collected at baseline, immediately after each treatment administration, and at six time points post-administration, with the last sample acquired when participants came out of the scanner at about 115 min post-administration (Figure 6). Saliva samples were acquired at baseline and together with the last blood sample. For the purposes of this report, we use the plasmatic and salivary oxytocin measurements that were obtained at baseline and at 115 min after the end of our last treatment administration (this means that our post-administration samples were collected 115 min after the intranasal administrations and 120 mins after the intravenous administration of oxytocin). The full time course of changes in plasmatic oxytocin after the administration of intranasal and intravenous oxytocin in this study has been reported elsewhere (Martins et al., 2020).

Schematic representation of the design of study A.

All subjects received first an administration of intranasal placebo - either by spray or nebuliser, then an intravenous administration of oxytocin (10 IU)/saline and then an intranasal administration of oxytocin (40 IU)/placebo, either by spray or nebuliser. Following drug administration, participants were placed in a Magnetic Resonance Imaging scanner for eight resting arterial spinal labeling (ASL) regional blood flow images of the brain and one resting BOLD fMRI scan. Saliva samples were collected before any drug administration (baseline) and after the scanning session (at 115 min after our last treatment administration). Plasma samples were collected before any drug administration, after any administered drug and then at several time-points during scanning session. For the current study, only time-points where saliva and plasma were concomitantly collected were considered – baseline and after scanning session. Detailed plasmatic pharmacokinetics of each route/method have been presented elsewhere (Martins et al., 2020). Adm. – administration; min – minutes; IN – Spray; NB – Nebuliser; IV – Intravenous.

All visits were conducted during the morning to avoid the potential confounding of circadian variations in oxytocin levels (Amico et al., 1989; Reppert et al., 1984). In addition, we also made sure that each participant was tested at approximately the same time across all four visits (all participants were tested in sessions with less than one hour difference in their onset time, except for one participant where the difference in the onset of one session compared to the other three sessions was 1.5 hr). All visits were identical in structure (duration ~3.5 hr). Upon arrival, participants gave consent and completed the required questionnaires. We then fitted an intravenous cannula on each arm of our participants (one for the intravenous administration and another for blood-sampling). Treatment was administered according to the study protocol (Figure 6). At the end of the treatment administration, participants were taken to an MRI scanner where we obtained a number of resting state and structural scans over the course of the next 2 hr.

Dataset B

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Participants were recruited to participate in a study examining the effects of MDMA on social cognition (Gabay et al., 2019). Briefly, baseline blood samples were obtained 15 min before MDMA/placebo administration on two separate occasions, spaced on average 9.30 days apart (SD = 5.70 days; range: 7–31 days).

Saliva and plasma collections

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Blood was collected in 5 ml ethylenediaminetetraacetic acid vacutainers (Kabe EDTA tubes 078001), placed in iced water and centrifuged at 1300 × g for 10 min at 4°C within 20 min of collection and then 0.5 ml of plasma was immediately pipetted into 2 ml Eppendorf vials. Samples were immediately stored −80°C until analysis. Saliva samples were collected using a salivette (Sarstedt 51.1534.500). Participants were instructed to place the swab from the Salivette kit in their mouth and chew it gently for 1 min to soak as much saliva as possible. After this, the swab was then returned back to the Salivette, centrifuged, 0.5 ml of saliva was aliquoted to 1.5 ml Eppendorf vials and then stored in the same manner as blood samples. Salivettes allow for a collection of mean saliva volumes in the range of 1.1 ± 0.3 ml (according to the manufacturer); high recovery of the concentrations of small peptides in saliva are consistently achieved when the sampled volumes are larger than 0.25 ml (Gröschl et al., 2008; Harmon et al., 2007). For both saliva and plasma, we followed the RIAgnosis standard operating procedures.

We followed this strict protocol, putting all samples in iced water until centrifugation with immediate storage at −80°C until analysis to minimize the impact putative differences in degradation of the peptide related to differences in the processing of the samples might have on the reliability of the estimated concentrations of oxytocin. Minimizing the time-interval samples were kept in the collection devices also allowed us to keep potential absorption to the walls of these recipients to a minimum (Gröschl et al., 2008).

Quantification of oxytocin in plasma and saliva samples

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For both datasets, plasma and saliva oxytocin levels were analysed by a third party (RIAgnosis, Munich, Germany) using a Radioimmunoassay (RIA), as previously described (Kagerbauer et al., 2013). Plasma samples were extracted before quantification. Saliva samples were not extracted prior to quantification since unpublished data from RIAgnosis found no differences in oxytocin concentrations between extracted and simply evaporated saliva samples. RIA has been previously standardised and validated and represents the gold-standard for oxytocin measurement in biological fluids (Kagerbauer et al., 2013; Landgraf, 1981; Landgraf and Günther, 1983; Landgraf et al., 1982a; Landgraf et al., 1983; Landgraf et al., 1982b; Martin et al., 2014). The detection limit is in the 0.1–0.5 pg/sample range, depending on the age of the tracer. Cross-reactivity with vasopressin, ring moieties and terminal tripeptides of both oxytocin and vasopressin and a wide variety of peptides comprising 3 (alpha-melanocyte-stimulating hormone) up to 41 (corticotrophin-releasing factor) amino acids are <0.7% throughout. The intra- and inter-assay variabilities are <10% (Kagerbauer et al., 2013).

Dataset A

Between-visits reliability analysis

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Reliability refers to the reproducibility of values of a measurement in repeated trials on the same individuals (Hopkins, 2000). Reliability can be quantified using two sets of metrics providing complementary information: absolute and relative reliability. Absolute reliability is the degree to which repeated measurements within the same subject vary over time (Hopkins, 2000). Relative reliability is the degree to which individuals maintain their position in a sample of subjects measured over time (Hopkins, 2000). Absolute and relative reliability of plasma and salivary oxytocin measurements were estimated using the within-subject CV and the ICC, respectively. ICC was estimated in a two-way mixed model, single measures, absolute agreement (Koo and Li, 2016). We first estimated the reliability across all four sessions, and subsequently for each pair of visits to assess if time-interval between sample collections may impact on reliability indexes estimation. Only participants presenting baseline measures across all four sessions were included in the reliability analysis, as previously suggested (Cuesta Izquierdo and Fonseca Pedrero, 2014).

Since there was considerable variability in the time-interval between visits across participants, we conducted a sensitivity analysis where we repeated our reliability analysis focusing on 15 pairs of consecutive measures that were collected with an exact time interval of 7 days between visits in 15 participants. Here, we recalculated the ICC and CV on this subset of our initial sample, using the approach described above. We also investigated whether within-participant variance in the time interval between sample acquisitions could predict within-participant variance in oxytocin concentrations across participants, using Spearman correlations.

Correlations are often used as an index for reliability, even though they cannot provide information about the absolute agreement of two sets of measurements (Qin et al., 2019). Hence, to facilitate comparisons with previous reports, we also calculated Pearson’s correlation coefficients, with bootstrapping (1000 samples), to evaluate correlations between baseline concentrations of oxytocin for each pair of visits. The results of this analysis are presented below in the Table S1 and Figure S2.

Within-visit reliability analysis (placebo visit)

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To investigate the reliability of salivary and plasmatic oxytocin concentration within the same visit, we calculated the ICC and CV as described above for two samples acquired before any treatment administration and the intravenous infusion of saline during the placebo session. These samples where acquired with an approximate 15 min interval in between them.

Mean concentrations across visits

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Mean concentrations of saliva and plasma oxytocin across the four visits were compared using repeated-measures one-way analysis of variance.

Treatment effects

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The effect of treatment on blood/saliva oxytocin concentration were assessed using a 4 × 2 repeated-measures two-way analysis of variance Treatment (four levels: Spray, Nebuliser, Intravenous and Placebo) × Time (two levels: Baseline and post-administration). Post-hoc comparisons to clarify a significant interaction were corrected for multiple comparisons following the Tukey procedure.

Association between salivary and plasmatic oxytocin levels

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We assessed correlations between salivary and plasmatic concentrations of oxytocin sampled at baseline and post-administration. For the baseline measurements, we pooled data across treatment levels because there were no differences between groups on mean baseline concentrations of oxytocin. To account for the non-independence among the four data points within each subject, we used multilevel correlation, where we modeled participant as a random effect. For the post-administration measurements, we calculated Pearson’s correlation coefficient for each treatment level separately because group differences in mean scores on these measures might result in illusory correlations if the drug and placebo samples were pooled together (Paloyelis et al., 2010). As a final sanity check, we also investigated correlations between the changes from baseline to post-administration in saliva and plasma in each of our treatment conditions separately. Since our sample was relatively small and therefore the lack of significant correlations between salivary and plasmatic oxytocin could simply reflect lack of sensitivity, we followed this frequentist correlation analysis with Bayesian statistics to quantify relative evidence for both the null and alternative hypotheses.

Outliers and missing values

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Salivary oxytocin concentrations were missing for three participants, and plasmatic oxytocin concentration for one participant. One measure of baseline oxytocin in saliva and two post-administration measures in the nebuliser condition were discarded after they had been identified as outliers. Outliers were identified using the outlier labelling rule (Kwak and Kim, 2017); this means that a data point was identified as an outlier if it was more than 1.5 x interquartile range above the third quartile or below the first quartile. A total of 13 and 16 participants were included in the reliability analysis of salivary and plasmatic oxytocin, respectively.

Dataset B

Mean concentrations across visits

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Mean concentrations of plasma oxytocin across the two visits were compared using a paired T-test.

Reliability analysis

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Absolute and relative reliability of plasma oxytocin measurements were analysed for the two baseline measures obtained from each of the two visits, following the methods described for dataset A.

Outliers and missing values

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There were no missing values. One baseline measure for one of the visits was discarded after being identified as an outlier. A total of 19 participants were included in the reliability analysis.

Increasing the number of observations per individual and averaging across several samples collected on different occasions is an approach commonly used to control within-individual variation and maximize reliability (Walker, 2008). Hence, we expanded our reliability analysis by asking how many additional measures of the same individual would be theoretically required to achieve different levels of reliability of a hypothetical averaged measure, based on the initial reliabilities estimated in the studies A and B. This number was calculated using the Spearman-Brown prediction formula (de Vet et al., 2017). For these calculations, we considered cut-offs of ICC = 0.50 (fair reliability), 0.70 (moderate), and 0.80 (good) as suggested by Koo and Li, 2016.

Statistical analysis

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All statistical analyses were conducted on log-transformed oxytocin concentrations given the deviations of these measurements from a Gaussian distribution. The statistical analysis investigating treatment effects on salivary and plasmatic oxytocin were performed using SPSS (version 24, IBM, Armonk, NY, USA). The frequentist and Bayesian correlations were implemented in the correlation package from R (version 3.5.3), using bootstrapping 1000 samples. For the bayesian correlations, we used beta priors’ distributions centred around zero, with a width parameter of 1. An increase in Bayes Factor (BF) in our analyses corresponds to an increase in evidence in favor of the null hypothesis. To interpret BF, we used the Lee and Wagenmakers’ classification scheme (Lee MD, 2014): BF <1/10, strong evidence for alternative hypothesis; 1/10 < BF < 1/3, moderate evidence for alternative hypothesis; 1/3 < BF < 1, anecdotal evidence for alternative hypothesis; BF >1, anecdotal evidence for the null hypothesis; 3 < BF < 10, moderate evidence for the null hypothesis; BF >10, strong evidence for the null hypothesis. Figures were produced using the ggplot package from R (version 3.5.3). p<0.05 (two-tailed) was set as threshold of statistical significance for all analyses.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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Decision letter

  1. Joseph G Gleeson
    Reviewing Editor; Howard Hughes Medical Institute, The Rockefeller University, United States
  2. Christian Büchel
    Senior Editor; University Medical Center Hamburg-Eppendorf, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Reviewers concurred that a single peripheral measurements of oxytocin at baseline may not provide valid trait markers of the physiology of the oxytocin system. The increases in salivary oxytocin observed after intranasal oxytocin most likely reflect unabsorbed peptide and should not be used by the field to predict treatment effects. eLife and reviewers appreciated the careful attention to methodology, which could influence interpretation or reinterpretation of many studies that rely on salivary and plasmatic oxytocin measurements.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Salivary and plasmatic oxytocin are not reliable biomarkers of the physiology of the oxytocin system in Humans" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The strengths of the study are the findings that a single oxytocin level measured from saliva or plasma is not meaningful in the way that the field might currently be measuring. The reviewers appreciated this finding, and the careful attention to detail, but felt that the results fell short of the level of insight required to meet the threshold for publication in eLife. In the setting where eLife decides not to proceed to publication, the reviews are returned in their unedited format for your benefit.

Reviewer #1:

This article describes the investigation of a valuable research question, given the interest in using salivary oxytocin measures as a proxy of oxytocin system activity. A strength of the study is the use of two independent datasets and the comparison between intranasal and intravenous administration. The authors report poor reliability for measuring salivary oxytocin across visits, that intravenous delivery does not increase concentrations, and that salivary and blood plasma concentrations are not correlated.

Introduction: While it's true that saliva collection provides logistical advantages, there are also measurement advantages (e.g., relatively clean matrix) that are summarised in the MacLean et al., 2019 study, which has already been cited.

It is important to note that the 1IU intravenous dose in this study led to equivalent concentrations in blood compared to intranasal administration

Materials and methods: When using both ELISA and HPLC-MS, extracted and unextracted samples are correlated when measuring oxytocin concentrations in saliva, at least in dogs (https://doi.org/10.1016/j.jneumeth.2017.08.033)

Statistical reporting: I ran the article through statcheck R package (a web version is also available) and found a number of inconsistencies with the reported statistics and their p values. For example, the authors reported: t(123) = 1.54, p = 0.41, but this should yield a p value of 0.13. The authors should do the same and fix these errors

Results: The confidence intervals for these correlations should be reported

Discussion, “Our observation of poor reliability for single measurements of plasma and saliva oxytocin raises questions about the interpretation of previous evidence seeking to associate single measurements of baseline oxytocin with individual differences in a range of neuro-behavioural or clinical traits…”: This is an important point, but it's important to note that the vast majority of these studies use plasma or saliva measures. Perhaps CSF measures are more reliable, but the question wasn't assessed in the present study, and I'm not sure if anyone has looked at this question.

Discussion final paragraph: I broadly agree with this conclusion, but it should be added that "single measurements of baseline levels of endogenous oxytocin in saliva and plasma are not stable under typical laboratory conditions" Perhaps these measures can be more stable using other means (i.e., better standardising collection conditions). But the fact remains, under typical conditions these measures do not demonstrate reliability

Reviewer #2:

To test questions whether salivary and plasmatic oxytocin at baseline reflect the physiology of the oxytocin system, and whether salivary oxytocin index its plasma levels, the authors quantified baseline plasmatic and/or salivary oxytocin using radioimmunoassay from two independent datasets. Dataset A comprised 17 healthy men sampled on four occasions approximately at weekly intervals. In the dataset A, oxytocin was administered intravenously and intranasally in a triple dummy, within-subject, placebo-controlled design and compared baseline levels and the effects of routes of administration. With dataset A, whether salivary oxytocin can predict plasmatic oxytocin at baseline and after intranasal and intravenous administrations of oxytocin were also tested. Dataset B comprised baseline plasma oxytocin levels collected from 20 healthy men sampled on two separate occasions. In both datasets, single measurements of plasmatic and salivary oxytocin showed insufficient reliability across visits (Intra-class correlation coefficient: 0.23-0.80; mean CV: 31-63%). Salivary oxytocin was increased after intranasal administration of oxytocin (40 IU), but intravenous administration (10 IU) does not significantly changes. Saliva and plasma oxytocin did not correlate at baseline or after administration of exogenous oxytocin (p>0.18). The authors suggest that the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid biomarkers of the physiology of the oxytocin system is questionable in men. Furthermore, they suggest that saliva oxytocin is a weak surrogate for plasma oxytocin and that the increases in saliva oxytocin observed after intranasal oxytocin most likely reflect unabsorbed peptide and should not be used to predict treatment effects.

The current study tested research questions relevant for the study field. The analyses in two independent datasets with different routes of oxytocin administrations is the strength of current study. However, the limited novelty of findings and several limitations are noticed in the current report as described below.

1) Previous study with similar results has already revealed that saliva oxytocin is a weak surrogate for plasmatic oxytocin, and increases in salivary oxytocin after the intranasal administration of exogenous oxytocin most likely represent drip-down transport from the nasal to the oral cavity and not systemic absorption (Quintana et al., 2018). Therefore, the novelty of current findings is limited. The authors should more clearly state the novelty of current results and the replication of previous findings.

2) As authors discussed in the limitation section of Discussion, the current study has several limitations such as analyses only in male participants and non-optimized timing of collection of saliva and blood due to the other experiments. These limitations are understandable, because the current study was the second analyses on the data of the other studies with the different aims. However, these limitations significantly limit the interpretations of the findings.

3) As reported the Materials and methods, the dataset A comprises administrations approximately 40 IU of intranasal oxytocin and 10 IU on intravenous. The rationale to set these doses should be described. Since the 40IU is different from 24 IU which is employed in most of the previous publications in the research filed, potential influence associated with the doses should be tested and discussed.

4) It is difficult to understand that no significant elevations in plasma oxytocin levels were observed after intranasal spray or nebuliser of oxytocin. From Figure 4A, the differences between levels at baseline and post administration are similar between nebuliser, spray, and placebo. Please discuss the potential interpretation on this result.

5) The reason why not to employ any correction for multipole comparisons in the statistical analyses should be clarified.

Reviewer #3:

Baseline samples of salivary and plasma oxytocin were assessed in 13, respectively, 16 participants, to assess intra-individual reliability across four time points (separated by approximately 8 days). The main results indicate that, while as a group, average salivary and plasma samples were not significantly different across time points, within-subject coefficient of variation (CV) and intra-class correlation coefficient (ICC) showed poor absolute and relative reliability of plasma and salivary oxytocin measurements over time. Also no association was established between plasma and salivary levels, either at baseline or after administration of oxytocin (either intranasally, or intravenously). Further, salivary/ plasma oxytocin was only enhanced after intranasal, respectively intravenous administration.

While the overall multi-session design seems solid, sample collections were performed in the context of larger projects and therefore there appear to be several limitations that reduce the robustness of the presented results and consequently the formulated conclusions.

General comments

1) A main conclusion of the current work is that “single measures of baseline oxytocin concentrations in saliva and plasma are not stable within the same individual”. It seems however that the study did not adhere to a sufficiently rigorous approach to put forward this conclusion. It lacks a control for several important factors, such as timing of the day at which saliva/ plasma samples were obtained, as well as sample volume.

Particularly while it is indicated that all visits were identical in structure, important information is missing with regard to whether or not sampling took place consistently at a particular point of time each day, to minimize the influence of circadian rhythm. Without this information it is not possible to draw any firm conclusions on the nature of the intra-individual variability as demonstrated in the salivary and plasma sampling.

Correspondingly, a deeper discussion is needed on the reason why ICC's were considerably variable across pairs of assessment sessions, with some pairs yielding good reliability, whereas others yielded (very) poor reliability. More detailed descriptions regarding sampling procedures (timing and sampling intervals) are necessary. Also, more information is needed on the volume of saliva collected at each session, to control for possible dilution effects.

2) It is indicated that the initial sample would allow to detect intra-class correlation coefficients (ICC) of at least 0.70 (moderate reliability) with 80% of power. Is this still the case after the drop-outs/ outlier removals? Since the main conclusions of the work rely on negative results (conclusions drawn from failures to reject the null hypothesis) it is important to establish the risk for false negatives within a design that is possibly underpowered.

3) Did the authors also assess within-session reliability? For example, by assessing ICC between pre and post-measurements in the placebo session.

4) It is indicated that the intra-assay variability of the adopted radioimmunoassay constitutes <10%. Were analyses of the current study run on duplicate samples? Was intra-assay variability assessed directly within the current sample?

Introduction and Discussion

5) The Introduction and Discussion is missing a thorough overview of previous studies assessing intra-individual variability in oxytocin levels.

6) The paper misses a discussion of previous studies addressing links between salivary/ plasma levels and central oxytocin (e.g. in cerebrospinal fluid). I understand the claim that salivary oxytocin cannot be used to form an estimate of systemic absorption, although technically, a lack of a link between salivary and plasma levels, does not necessarily imply a lack of a relationship to e.g. central levels. The lack of effect is limited to this specific relationship.

Materials and methods

7) Related to the general comment, the variability in days between sessions is relatively high (average 8.80 days apart (SD 5.72; range 3-28). However, it appears that no explicit measures were taken to control the conducted analyses for this variability.

8) A rationale for the adopted dosing and timing (115 min post administration) of the sample extraction is missing. Additionally, it seems that intravenous administrations were always given second, whereas intranasal administrations were given third, with a small delay of approximately 5 min. Hence, it seems that the timing of 115 min post-administration is only accurate for the intranasal administration.

9) Since the ICC of baseline samples showed poor reliability, it seems suboptimal to pool across sessions for assessing the relationship between salivary and blood measurements. It should be possible to perform e.g. partial correlations on the actual scores, thereby correcting for the repeated measure (subject ID). Further, since the sample size is relatively small (13 subjects), it might be recommended to use non-parametric (e.g. Spearmann correlations) instead of Pearson. The additional reporting of the Bayes factor is appreciated; it is very informative.

10) Now, the authors only compared relationships between salivary and plasma levels, either at baseline or post administration. I'm wondering whether it would be interesting to explore relationships between pre-to-post change scores in salivary versus plasma measures.

11) Please provide more information on the outlier detection procedure (outlier labelling rule).

12) Please indicate how deviations from a Gaussian distribution were assessed.

Results

13) Please verify the degrees of freedom for the post-hoc tests performed to assess pre-post changes at each treatment level (e.g. baseline vs Post administration: Spray – t(122) = 7.06, p < 0.001). Why is this 122? Shouldn't this be a simple paired-sample t-test with 13 subjects?

Reviewer #3:

• I would omit references to “biomarkers” or “biomarkers of the physiology of the oxytocin system” (e.g. in the title). I'm not sure which previous studies have claimed this link.

• In the last paragraph of the Introduction, only reference is made to assessing the effect of intravenous administration, whereas the study addresses both intravenous and intranasal effects.

• In the Materials and methods it is stated that “participants had no history of psychiatric disorders or substance abuse”. How was this assessed? Did the authors adopt any particular questionnaire or scale?

• Please indicate explicitly the number of subjects involved in any of the (Pearson) correlation analyses e.g. assessing relationships between salivary and plasma measurements.

• In the Discussion, it is indicated that “the time interval between measurements do not seem to significantly impact on the reliability of baseline oxytocin”. This was not explicitly assessed.

• Abbreviations in figures need to be reported in full in the figure legend.

• Figure 2. It would be helpful to use the same range for the y-axis of the plasma assessments (in sample A and B).

• It would be recommended to use a consistent order for reporting the different treatment levels. Now the table in Figure 1 uses the order spray, IV, placebo, Nebulizer, whereas in Figure 2, this order is changed to IV, nebulizer, placebo, spray. I guess the most logic order would be spray, nebulizer, IV and placebo (as used in-text).

• It is unclear why the presentation modes for presenting the associations between salivary and plasma OT at baseline (Figure 4) and post-administration (Figure 5) are different. It would be informative to plot for both the regression lines and distribution plots.

• Supplementary figures need figure legends.

• I would recommend omitting the reporting of “dataset B” from the main manuscript, or only report it (briefly) as a secondary analysis, with some additional information in the supplements.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting the appeal for your article "Salivary and plasmatic oxytocin are not reliable biomarkers of the physiology of the oxytocin system in Humans" for consideration by eLife. The appeal has been reviewed by two of the original peer reviewers, and the evaluation has been overseen by Reviewing Editor Joseph Gleeson and Christian Büchel as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers are now more convinced by the data, and offer specific requests that, if completed, should allow the paper to proceed towards publication. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Reviewer #1:

This is a revision of an article that I previously reviewed investigating if single peripheral measurements of baseline oxytocin in saliva and plasma are reliable trait markers of the physiology of the oxytocin system in humans.

The paper has now improved and most of my original queries have now been satisfactorily addressed.

However, I still have one comment regarding the author's response to query #2 "It is important to note that the 1IU intravenous dose in this study led to equivalent concentrations in blood compared to intranasal administration": I now better understand the justification for using a 10IU dose (i.e., "we demonstrate that even when plasmatic levels of OT are maintained substantially increased throughout the observation interval, we cannot detect increases in salivary oxytocin". However, this should also be better emphasized in the manuscript.

Reviewer #3:

Overall, the authors were able to provide additional information and conduct additional analyses that provided solutions to several of the raised methodological concerns (e.g. regarding timing of saliva collection, within-session reliability, sample size/ power, and other statistical remarks). Some other methodological issues may still need further clarification however, such as the possible impact of variability in collected sample volume across sessions (at least for the salivary samples). Also, considering the main research question of the current study (reliability of oxytocin sampling), it would be recommended that a measure of intra-assay variability was available for the own sample collections. The efforts for accounting for the effect of variability in between-session intervals are appreciated. I'm wondering however, whether it would be possible to perform secondary analyses on the whole data set (rather than performing subset analyses) regressing out possible effects of variability in between-session intervals.

In general, I feel that the manuscript already improved significantly compared to the initial submission, increasing its potential for publication tremendously. However, some of the raised concerns regarding the relative novelty regarding the study design and conclusions may still remain, raising questions whether a more specialized journal may be more suitable for its publication.

https://doi.org/10.7554/eLife.62456.sa1

Author response

[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

The strengths of the study are the findings that a single oxytocin level measured from saliva or plasma is not meaningful in the way that the field might currently be measuring. The reviewers appreciated this finding, and the careful attention to detail, but felt that the results fell short of the level of insight required to meet the threshold for publication in eLife. In the setting where eLife decides not to proceed to publication, the reviews are returned in their unedited format for your benefit.

We would like to thank you for the careful consideration of our manuscript and the three reviewers for their insightful comments. Overall, it seems to us the reviewers and editorial team found that the topic of our manuscript makes a valuable addition to the field. We have analysed in detail all reviewers’ comments and note that, overall, they are positive, fair and constructive. However, reading through the reviewers’ feedback, we felt that as a result of our insufficient clarity in communicating certain aspects of our work, the novelty and robustness of our findings might have been misunderstood. In particular, the reviewers raised concerns about the fact that we were not sufficiently strict in controlling major sources of variability that could have affected the levels of oxytocin in saliva and plasma between sessions, such as time of the day the samples were collected. This was not the case, since we used a consistent protocol where we controlled for time of day, food and liquid ingestion and pre-processing time of the samples, to minimize any potential influence from known sources of variability in oxytocin release to the periphery. The reviewers also raised concerns about reporting erroneous p-values and not having used correction for multiple comparisons where required, which as we explain in our responses below are not accurate and simply reflect lack of clarity in our report.

Prompted by the reviewers, we have now conducted some further analyses that strengthen the manuscript and add valuable insight into the interpretation of the data. For instance, we now show that the within-session reliability of salivary and plasmatic oxytocin in the placebo session is excellent. This strengthens our findings of low reliability across different days by suggesting that such poor reliability cannot simply be explained by measurement error or variation in the measurement conditions across sessions. We also conducted further analyses that discount variability in the time-interval between visits as a potential factor contributing to the low reliabilities we report in the manuscript.

Reviewer #1:

This article describes the investigation of a valuable research question, given the interest in using salivary oxytocin measures as a proxy of oxytocin system activity. A strength of the study is the use of two independent datasets and the comparison between intranasal and intravenous administration. The authors report poor reliability for measuring salivary oxytocin across visits, that intravenous delivery does not increase concentrations, and that salivary and blood plasma concentrations are not correlated.

Introduction: While it's true that saliva collection provides logistical advantages, there are also measurement advantages (e.g., relatively clean matrix) that are summarised in the MacLean et al., 2019, study, which has already been cited.

Thanks for the suggestion. We added this advantage:

“Compared to blood sampling, saliva collection presents several logistical and measurement advantages (i.e. relatively clean matrix)(1).”

It is important to note that the 1IU intravenous dose in this study led to equivalent concentrations in blood compared to intranasal administration

The reviewer is right that 10 IU (over 10min) in our case increased the concentrations of plasmatic oxytocin beyond those observed for the spray or nebuliser (we reported the full time-course of variations in plasmatic oxytocin in another manuscript we published earlier this year)(2). This was an intentional aspect of our study design. We decided to use the highest intravenous dose (at the highest rate of 1IU/min) that we could get permission to administer safely in healthy volunteers as a proof of concept, so as to achieve a robust and prolonged increase in plasmatic oxytocin over the course of our full testing session. In this manner, we demonstrate that even when plasmatic levels of OT are maintained substantially increased throughout the observation interval, we cannot detect increases in salivary oxytocin. In this aspect, we believe that our manuscript goes one step beyond the important findings described in of Quintana et al. 2018(3), showing that this phenomenon is not linked to dosage (or to amount of increase in plasmatic levels of exogenous OT), as far as we can determine given the current safety standards for the administration of OT IV.

Please see also response to reviewer 2, point 1.

Materials and methods: When using both ELISA and HPLC-MS, extracted and unextracted samples are correlated when measuring oxytocin concentrations in saliva, at least in dogs (https://doi.org/10.1016/j.jneumeth.2017.08.033)

Thanks for pointing out this study. Indeed, in this specific study the authors found correlations between extracted and unextracted saliva samples. Such associations in humans have nevertheless been rare. In humans, the body of evidence suggests that the measurements obtained when comparing extracted samples to unextracted samples, or when comparing samples obtained using different methods of quantification (for instance, ELISA versus radioimmunoassay), do not correlate or show very low correlations (4, 5). Furthermore, most ELISA kits and HPLC-MS protocols to measure oxytocin have so far fallen short on sensitivity to detect the typical concentrations observed in humans at baseline (0-10pg/ml)(6). The current gold-standard method for quantifying oxytocin in biological fluids is the radioimmunoassay we used in this study(4). This method has shown superior sensitivity and specificity when compared to other quantification methods, when combined with extracted samples; therefore, it was our primary choice. We now highlight this advantage in the revised version of the manuscript more explicitly.

“For all analyses, we followed current gold-standard practices in the field and assayed oxytocin concentrations using radioimmunoassay in extracted samples, which has shown superior sensitivity and specificity when compared to other quantification methods(7).”

Statistical reporting: I ran the article through statcheck R package (a web version is also available) and found a number of inconsistencies with the reported statistics and their p values. For example, the authors reported: t(123) = 1.54, p = 0.41, but this should yield a p value of 0.13. The authors should do the same and fix these errors

Thanks very much for taking the time to check our statistical reporting thoroughly. We apologize if we were not sufficiently clear in the previous version of the manuscript, but the p-values we reported are corrected for multiple comparisons using Tukey correction. Currently, statcheck can only evaluate inconsistencies when the results are reported in the standard APA style and does not take into consideration corrections for multiple comparisons of any kind. We did check all of our statistical reporting and the p-values and correspondent statistics are correct (we only corrected an inadvertent error in reporting the degrees of freedom for these tests). In any case, we have now clarified in the manuscript when the reported p-values have been adjusted for multiple comparison to avoid any further confusion.

Results: The confidence intervals for these correlations should be reported

We have now added the confidence intervals, estimated using bootstrapping, in our Results section.

Discussion, “Our observation of poor reliability for single measurements of plasma and saliva oxytocin raises questions about the interpretation of previous evidence seeking to associate single measurements of baseline oxytocin with individual differences in a range of neuro-behavioural or clinical traits…”: This is an important point, but it's important to note that the vast majority of these studies use plasma or saliva measures. Perhaps CSF measures are more reliable, but the question wasn't assessed in the present study, and I'm not sure if anyone has looked at this question.

We are not aware of any study evaluating the stability of measurements of oxytocin in the CSF. Indeed, there are only a few studies sampling CSF to measure oxytocin in clinical patients and it is unlikely that CSF will become a widely used fluid to measure oxytocin in humans, given the invasiveness of the procedure to obtain CSF samples. Here, we wanted to refer specifically to saliva and plasma, which remain as the most popular options for measuring oxytocin in humans and which we investigated specifically in the current study. We have changed the text accordingly for clarity.

“Our data poses questions about the interpretation of previous evidence seeking to associate single measurements of baseline oxytocin in saliva and plasma with individual differences in a range of neuro-behavioural or clinical traits.”

Discussion final paragraph: I broadly agree with this conclusion, but it should be added that "single measurements of baseline levels of endogenous oxytocin in saliva and plasma are not stable under typical laboratory conditions" Perhaps these measures can be more stable using other means (i.e., better standardising collection conditions). But the fact remains, under typical conditions these measures do not demonstrate reliability

Thanks for the suggestion. We have revised the text accordingly throughout the manuscript (examples below). Our study is a pharmacological study, which means that it is conducted in a highly controlled setting and adheres to strict protocols (i.e. we tested participants at the same time of the day, we instructed participants to abstain from alcohol and heavy exercise for 24 h and from any beverage or food for 2 h before scanning). These exclusion criteria were stricter than those applied in a large number of studies sampling saliva and plasma for measuring oxytocin for the purposes estimating possible associations with various traits associating. Most of these studies do not control, for instance, for fluid or food ingestion. Therefore, we expected our reliability calculations to represent an optimistic estimate of the reliabilities of the salivary and plasmatic oxytocin concentration used in most studies.

For now, it remains unclear to us what factors might be driving the within-subject variability in salivary and plasmatic concentrations we report in this study. Thanks to reviewer 3, we are now confident that this is unlikely to represent measurement error (see response to reviewer 3, point 3).

“Here, we aimed to characterize the reliability of both salivary and plasmatic single measures of basal oxytocin in two independent datasets, to gain insight about their stability in typical laboratory conditions and their validity as trait markers for the physiology of the oxytocin system in humans.”

“In summary, single measurements of baseline levels of endogenous oxytocin in saliva and plasma as obtained in typical laboratory conditions are not stable and therefore their validity as trait markers of the physiology of the oxytocin system is questionable.”

Reviewer #2:

To test questions whether salivary and plasmatic oxytocin at baseline reflect the physiology of the oxytocin system, and whether salivary oxytocin index its plasma levels, the authors quantified baseline plasmatic and/or salivary oxytocin using radioimmunoassay from two independent datasets. Dataset A comprised 17 healthy men sampled on four occasions approximately at weekly intervals. In the dataset A, oxytocin was administered intravenously and intranasally in a triple dummy, within-subject, placebo-controlled design and compared baseline levels and the effects of routes of administration. With dataset A, whether salivary oxytocin can predict plasmatic oxytocin at baseline and after intranasal and intravenous administrations of oxytocin were also tested. Dataset B comprised baseline plasma oxytocin levels collected from 20 healthy men sampled on two separate occasions. In both datasets, single measurements of plasmatic and salivary oxytocin showed insufficient reliability across visits (Intra-class correlation coefficient: 0.23-0.80; mean CV: 31-63%). Salivary oxytocin was increased after intranasal administration of oxytocin (40 IU), but intravenous administration (10 IU) does not significantly changes. Saliva and plasma oxytocin did not correlate at baseline or after administration of exogenous oxytocin (p>0.18). The authors suggest that the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid biomarkers of the physiology of the oxytocin system is questionable in men. Furthermore, they suggest that saliva oxytocin is a weak surrogate for plasma oxytocin and that the increases in saliva oxytocin observed after intranasal oxytocin most likely reflect unabsorbed peptide and should not be used to predict treatment effects.

The current study tested research questions relevant for the study field. The analyses in two independent datasets with different routes of oxytocin administrations is the strength of current study. However, the limited novelty of findings and several limitations are noticed in the current report as described below.

1) Previous study with similar results has already revealed that saliva oxytocin is a weak surrogate for plasmatic oxytocin, and increases in salivary oxytocin after the intranasal administration of exogenous oxytocin most likely represent drip-down transport from the nasal to the oral cavity and not systemic absorption (Quintana et al., 2018). Therefore, the novelty of current findings is limited. The authors should more clearly state the novelty of current results and the replication of previous findings.

We apologize for not describing the novelty and impact of our findings with sufficient clarity, and thanks for the opportunity to do so. Our study had two major goals. The first was to investigate whether single measurements of salivary and plasmatic concentrations of oxytocin can be reliably estimated within the same individual when collected at baseline conditions (i.e. without any experimental manipulation). As the reviewer highlighted, this is an important methodological question given the wide use of these measurements in a large and increasing number of studies to establish associations between the physiology of the oxytocin system and a number of brain and behavioural phenotypes in both clinical and non-clinical samples. However, to our knowledge, no previous study has appropriately conducted a thorough investigation of the reliability of these measurements (see also response to reviewer 3, point 5). Thanks to our study, we now know that when single measurements are collected at baseline, salivary and plasmatic oxytocin cannot provide a sufficiently stable trait marker of the physiology of the oxytocin system in humans. As we highlight in the manuscript, this finding should deter the field from making strong claims based exclusively on associations of phenotypes with single measurements of peripheral oxytocin concentrations. Furthermore, our study also describes two very concrete implications of our findings which we believe are very important for the field. First, if baseline level of OT is to be used as a trait marker, future studies should, as much as possible, rely on repeated measures within the same participant but collected on different days to maximize reliability. Second, this less than perfect reliability should be taken into consideration when calculating the sizes of the samples needed to detect a certain effect, if it exists, with sufficient statistical power.

The second goal of our study was, as pointed out by the reviewer, to revisit the findings of Quintana et al., 2018(3), but this time with two major design modifications which could strengthen the conclusions from that study.

The first modification was the dose of intravenous oxytocin administered, which was considerably higher (see response to reviewer 1, point 2). The administration of a higher dose that resulted in substantial and sustained increases in plasmatic oxytocin throughout the two hours observation period can only strengthen the previous conclusion that increases in plasmatic oxytocin cannot be detected in salivary measurements, and that this is not a matter of dose (as far as we can ascertain by administering the maximum intravenous dose we could safely administer in healthy volunteers). We believe that this is an important addition to the literature.

The second modification regarded the choice of the method we used to quantify oxytocin. In this study, we used radioimmunoassay, which is superior to ELISA in sensitivity and hence more appropriate to measure the low concentrations of oxytocin in saliva and plasma typically detected in humans at baseline conditions (1-10 pg/ml; for most individuals 1-5 pg/ml)(6). For instance, in Quintana et al., 2018(3) the limitations in the sensitivity of the ELISA kit used led the authors to discard around 50% of the collected saliva samples. Hence, our study replicates and extends the previous findings from Quintana et al., 2018 in important ways, demonstrating that the lack of an association between increases plasmatic oxytocin and salivary measurements is not limited by the dose of intravenous oxytocin administered or limitations of the sensitivity of the method used to quantify oxytocin.

We have now made the novelty and contribution of our work more explicit:

“Currently, we lack robust evidence that single measures of endogenous oxytocin in saliva and plasma at rest are stable enough to provide a valid trait marker of the activity of the oxytocin system in healthy individuals. […] Such evidence is urgently required, given reports that plasma and saliva levels of oxytocin are frequently altered during neuropsychiatric illness and that they co-vary with clinical aspects of disease(13).”

“Our findings were not consistent with these expectations. We could replicate previous evidence that intravenous oxytocin does not increase salivary oxytocin(3) and extended it by showing that the lack of increase in salivary oxytocin is not limited to the specific low dose of intravenous OT that was previously used (1IU) and that it is not driven by the insufficient sensitivity of the OT measurement method (which had resulted in more than 50% of the saliva samples being discarded in the previous study(3).”

2) As authors discussed in the limitation section of Discussion, the current study has several limitations such as analyses only in male participants and non-optimized timing of collection of saliva and blood due to the other experiments. These limitations are understandable, because the current study was the second analyses on the data of the other studies with the different aims. However, these limitations significantly limit the interpretations of the findings.

Here, we would like to highlight two aspects. First, most studies in the field are indeed conducted in men to avoid potential confounding from fluctuations in oxytocin concentrations across the menstrual cycle in women. Therefore, our study is representative of the typical samples used in most human studies. Second, we did not optimize our study to collect repeated samples of saliva. Indeed, it would have been interesting to describe the full-time course of variations of oxytocin concentrations in saliva after intranasal and intravenous administration. However, this does not detract the importance of our findings in respect to our first aim (which was our main goal).

We agree with the reviewer though that it is at least theoretically possible that we could have missed the window for increases in salivary oxytocin after intravenous oxytocin if it existed, given that we only sampled one post-administration time-point. However, we believe this was unlikely for one reason. Despite the sustained increase (throughout the two-hour observation interval) in plasmatic oxytocin following the intravenous administration of oxytocin, we observed no increase in salivary oxytocin post-dosing (at ~115 min). Unless the half-life of oxytocin is shorter in saliva than in the blood (which we do not know yet), we expected the levels of salivary oxytocin to mirror the changes in plasma – potentially with a slight delay given the time that it might take for oxytocin concentrations to build up in saliva through ultrafiltration from the blood, but this was not the case. Most likely the half-life of oxytocin in the saliva is not shorter than in the blood, since a previous study found increased concentrations of oxytocin in saliva up to 7h after administration of intranasal oxytocin (as the reviewer pointed out below, in our study we no longer could detect significant increases in plasmatic oxytocin after the intranasal administration of 40 IU with two different methods at around 115 mins post-administration). Therefore, while we acknowledge these limitations we also believe they do not detract from the importance of our main findings and the potential they hold to influence the field towards a more rigorous use of these measurements. Please see below for the implemented changes in the text.

“It is possible that we may have missed peak increases in saliva oxytocin after the intravenous administration of exogenous oxytocin if they occurred between treatment administration and post-administration sampling. This is unlikely given that the dose we administered intravenously resulted in sustained increases in plasmatic oxytocin over the course of two hours. Unless the half-life of oxytocin in saliva is much shorter than in the plasma, it would be surprising to not find any increases in salivary oxytocin after intravenous oxytocin given that concentrations of oxytocin in the plasma were still elevated at the specific time-point of our second saliva sample. Currently, we have no estimate for the half-life of oxytocin in saliva; however, given that previous studies have found evidence of increased salivary oxytocin after single intranasal administrations of 16IU and 24IU oxytocin up to seven hours post-administration(19), it is unlikely that the half-life of oxytocin is shorter in the saliva than in the plasma.”

3) As reported the Materials and methods, the dataset A comprises administrations approximately 40 IU of intranasal oxytocin and 10 IU on intravenous. The rationale to set these doses should be described. Since the 40IU is different from 24 IU which is employed in most of the previous publications in the research filed, potential influence associated with the doses should be tested and discussed.

Thank you for the opportunity to clarify this aspect of our work. With respect of our primary aims (to investigate whether single measurements of salivary and plasmatic oxytocin at baseline can be reliably measured within individuals across different days), the choice of doses is of course not relevant.

With respect to our secondary aim, namely, to investigate whether salivary oxytocin can be used to index concentrations of oxytocin in the plasma, particularly after the administration of synthetic oxytocin using the intranasal and intravenous routes, the administered doses are relevant.

The data reported here were collected as part of a larger project – which determined the choice of both intranasal and IV doses (2). As explained in our response to reviewer 1, point 2, the selection 10IU (over 10min) was the highest intravenous dose that we could get permission to administer safely in healthy volunteers as a proof of concept, so as to achieve a robust and prolonged increase in plasmatic oxytocin over the course of our full testing session. In this manner, we demonstrate that even when plasmatic levels of OT are maintained substantially increased throughout the observation interval, we cannot detect increases in salivary oxytocin.

Regarding the intranasal OT dose, it is worth noting that the 24 IU is indeed popular in oxytocin studies, but not exclusive, and generally the selection of dose in oxytocin studies has not been informed by detailed dose-response characterizations. Our choice of 40IU was made for the purposes of matching our previous work on the pharmacodynamics of OT in healthy volunteers(20), and is a dose we (21-29) and others (e.g. (30)) have commonly used with patients.

A potentially important implication if dose variations also imply variation in the total volume of liquid administered (as is usually the case with standard nasal sprays – but not with the nebuliser), then it is likely that the potential for drip-down might increase for higher volumes and decrease for lower volumes. As far as we know, no study has ever investigated the impact of administered volume on salivary oxytocin after the intranasal administration of synthetic oxytocin, but we agree this would be an important point to look at. We have now expanded our Discussion to accommodate this point.

“We expect this phenomenon to be particularly pronounced for higher administered volumes. Further studies should examine the impact of different administered volumes on increases in salivary oxytocin.”

4) It is difficult to understand that no significant elevations in plasma oxytocin levels were observed after intranasal spray or nebuliser of oxytocin. From Figure 4A, the differences between levels at baseline and post administration are similar between nebuliser, spray, and placebo. Please discuss the potential interpretation on this result.

The plasmatic concentrations of oxytocin we report in this study refer solely to the samples acquired at around 2h after the administration of intranasal oxytocin. We reported the full-time course of changes in plasmatic oxytocin in a paper published earlier this year(2) – which we now refer the reader to. We did find increases in plasmatic oxytocin after administration of oxytocin with the spray and nebuliser (around 3x the baseline concentrations) that did not differ between intranasal methods of administration. Plasmatic oxytocin reached a peak within 15 mins from the end of the intranasal administrations. Given the short half-life of oxytocin in the plasma, we believe it is not surprising that at 115 mins after the end of our last treatment administration the concentrations of oxytocin in the plasma are no longer different from the placebo condition.

“The full time course of changes in plasmatic oxytocin after the administration of intranasal and intravenous oxytocin in this study has been reported elsewhere(2).”

5) The reason why not to employ any correction for multipole comparisons in the statistical analyses should be clarified.

We apologize that this was not sufficiently clear, but we did correct for multiple testing using the Tukey procedure in our analyses investigating the effects of treatment on salivary and plasmatic oxytocin (this was described in Treatment effects). If the reviewer meant something else, we would be glad to follow any further advice on multiple testing correction he/she might have.

“Treatment effects: The effect of treatment on blood/saliva oxytocin concentration were assessed using a 4 x 2 repeated-measures two-way analysis of variance Treatment (four levels: Spray, Nebuliser, Intravenous and Placebo) x Time (two levels: Baseline and post-administration). Post-hoc comparisons to clarify a significant interaction were corrected for multiple comparisons following the Tukey procedure.”

Reviewer #3:

Baseline samples of salivary and plasma oxytocin were assessed in 13, respectively, 16 participants, to assess intra-individual reliability across four time points (separated by approximately 8 days). The main results indicate that, while as a group, average salivary and plasma samples were not significantly different across time points, within-subject coefficient of variation (CV) and intra-class correlation coefficient (ICC) showed poor absolute and relative reliability of plasma and salivary oxytocin measurements over time. Also no association was established between plasma and salivary levels, either at baseline or after administration of oxytocin (either intranasally, or intravenously). Further, salivary/ plasma oxytocin was only enhanced after intranasal, respectively intravenous administration.

While the overall multi-session design seems solid, sample collections were performed in the context of larger projects and therefore there appear to be several limitations that reduce the robustness of the presented results and consequently the formulated conclusions.

General comments

1) A main conclusion of the current work is that “single measures of baseline oxytocin concentrations in saliva and plasma are not stable within the same individual”. It seems however that the study did not adhere to a sufficiently rigorous approach to put forward this conclusion. It lacks a control for several important factors, such as timing of the day at which saliva/ plasma samples were obtained, as well as sample volume.

Particularly while it is indicated that all visits were identical in structure, important information is missing with regard to whether or not sampling took place consistently at a particular point of time each day, to minimize the influence of circadian rhythm. Without this information it is not possible to draw any firm conclusions on the nature of the intra-individual variability as demonstrated in the salivary and plasma sampling.

Thanks for pointing this out. Indeed, we were not sufficiently explicit on how strict we were in controlling for some potential sources of variability that could have contributed to the lack of reliability we report here. Our data was acquired in the context of two human pharmacological studies, which by design were strict on a number of aspects to minimize unwarranted noise. All participants were tested in the same period of the day (morning) to avoid the potential contribution of circadian fluctuations of oxytocin. In dataset A, we tried, as much as possible, to match the exact time participants were tested between visits, using the start time of the first visit as a reference. With the exception of one participant, where one session was conduct 1h and 30 mins later than the other three, all the remaining participants from study A were tested within 1h of the exact start time of session 1. Further, we also instructed participants to abstain from alcohol and heavy exercise for 24 h and from any beverage or food for 2 h before scanning. Hence, we believe our sampling protocol was strict enough to discard any potential contribution of major known sources of variability in oxytocin levels.

The reviewer also inquiries about the volume of the samples. For the plasma samples, we used a standardized protocol and collected the same blood volume in all participants, visits and time-points (1 EDTA tube of approximately 4 ml). The saliva samples were collected using Salivettes. Participants were instructed to place the swab from the Salivette kit in their mouth and chew it gently for 1 min to soak as much saliva as possible. After this, the swab was then returned back to the Salivette and centrifuged. In both cases, to avoid degradation of the peptide in the collected sample, we followed a strict protocol where all samples were put immediately in iced water until centrifugation, which happened within 20 mins of sample collection. Samples were then immediately stored at -80°C until analysis. Hence, differences in degradation of the peptide related to the processing of the sample are also unlikely to justify the poor reliabilities we report here.

For completeness, we have now added all of these further details to our Materials and methods section.

“All visits were conducted during the morning to avoid the potential confounding of circadian variations in oxytocin levels(31, 32). In addition, we also made sure that each participant was tested at approximately the same time across all four visits (all participants were tested in sessions with less than one hour difference in their onset time, except for one participant where the difference in the onset of one session compared to the other three sessions was 1.5h). “

“Blood was collected in ethylenediaminetetraacetic acid vacutainers (Kabe EDTA tubes 078001), placed in iced water and centrifuged at 1300 × g for 10 minutes at 4°C within 20 minutes of collection and then immediately pipetted into Eppendorf vials. Samples were immediately stored -80°C until analysis. […] We followed this strict protocol, putting all samples in iced water until centrifugation with immediate storage at -80°C until analysis to minimize the impact putative differences in degradation of the peptide related to differences in the processing of the samples might have on the reliability of the estimated concentrations of oxytocin.”

Correspondingly, a deeper discussion is needed on the reason why ICC's were considerably variable across pairs of assessment sessions, with some pairs yielding good reliability, whereas others yielded (very) poor reliability.

Currently we have no insightful hypothesis on why this could have been the case. Indeed, we found higher ICCs for only 2 out of 6 pairs of visits for the plasma. However, it is plausible that this might have occurred by chance. In any case, we should note that the 95% confidence intervals for the ICCs of our different pairs of samples overlap; this suggests that there is no evidence that the ICCs we estimated for the specific two pairs where we found higher reliabilities are significantly higher than those observed in the remaining pairs.

“If there are specific reasons explaining the higher reliability indices observed for the specific pairs of sessions, these reasons remain to be elucidated. However, it is not implausible that we might have found higher reliabilities for these specific two pairs by chance, since the 95% confidence intervals for the ICCs for all pairs of samples overlapped.”

More detailed descriptions regarding sampling procedures (timing and sampling intervals) are necessary. Also, more information is needed on the volume of saliva collected at each session, to control for possible dilution effects.

This information has been added to the revised version of the manuscript (please see response to your point number 1). As a further clarification, oxytocin concentrations were measured in plasma and saliva aliquots of 0.5 ml, following the standard operating procedures of RIAgnosis. This volume was used for all participants, sessions and time-points. Furthermore, for measuring cortisol, the salivettes were shown to allow for an almost 100% recovery, regardless of cortisol concentration, volume of the sample or method of quantification(33), suggesting that the sampling method is robust.

2) It is indicated that the initial sample would allow to detect intra-class correlation coefficients (ICC) of at least 0.70 (moderate reliability) with 80% of power. Is this still the case after the drop-outs/ outlier removals? Since the main conclusions of the work rely on negative results (conclusions drawn from failures to reject the null hypothesis) it is important to establish the risk for false negatives within a design that is possibly underpowered.

We understand the concern of the reviewer. However, according to the power calculations provided by Bujang and Baharum, 2017(34), the four repeated samples we collected in Dataset A would have allowed us to detect an ICC of 0.5 with 80% of statistical power even with only 13 subjects (which is the lowest sample size we used for the analysis on saliva in dataset A). The two samples we collected in Dataset B would allow us to detect an ICC of 0.6 with 80% of statistical power even with only 19 subjects. Hence, both datasets were powered to detect an ICC of 0.7 with acceptable power, if it existed, even after the exclusion of outliers.

3) Did the authors also assess within-session reliability? For example, by assessing ICC between pre and post-measurements in the placebo session.

Thanks for the suggestion. Indeed, we had not performed this analysis before but we agree it would be informative. We calculated the ICC and CV for the two samples acquired before any treatment administration and the intravenous infusion of saline during the placebo session. These samples where acquired with an approximate 15 min interval in between them. In this analysis, we found that the ICC was excellent 0.92 and the CV 20%. This additional analysis strengthens our findings by supporting the idea that our poor reliabilities across different days reflect true biological variability and cannot be attributed to measurement error. These new findings have now been included in the revised version of the manuscript.

Abstract

“Results: Single measurements of plasmatic and salivary oxytocin showed poor reliability across visits in both datasets. The reliability was excellent when samples were collected within 15 minutes from each other in the placebo visit.”

“Within-visit reliability analysis: To investigate the reliability of salivary and plasmatic oxytocin concentration within the same visit, we calculated the ICC and CV as described above for two samples acquired before any treatment administration and the intravenous infusion of saline during the placebo session. These samples where acquired with an approximate 15 minutes interval in between them.”

“Furthermore, in a further analysis assessing the within-session stability of plasmatic oxytocin using two measurements collected 15 minutes apart from each other in the placebo visit (one sample collected at baseline and the other after the intravenous administration of saline), we found excellent within-session reliability (ICC=0.92, CV=20%). Together, this suggests that the low reliability of endogenous oxytocin measurements across visits in the current study results from true intrinsic individual biological variability and not technical variability/error in the method used for oxytocin quantification.”

4) It is indicated that the intra-assay variability of the adopted radioimmunoassay constitutes <10%. Were analyses of the current study run on duplicate samples? Was intra-assay variability assessed directly within the current sample?

We reported the intra-assay variability determined by RIAgnosis during the development of this assay(35). This was not specifically assessed for the current study.

Introduction and Discussion

5) The Introduction and Discussion is missing a thorough overview of previous studies assessing intra-individual variability in oxytocin levels.

Thanks for the suggestion. We have now included in our Introduction/Discussion an overview of previous studies attempting to tackle this question, which unfortunately do not address this question with sufficient detail or using the appropriate methods and statistical analyses (see response to reviewer 2, point 1). Hence, from the available evidence, it is not possible to draw robust conclusions about the validity of concentrations of oxytocin in saliva and plasma as valid trait markers of the activity of the oxytocin system. With this manuscript, we hope we can prompt further discussion and guide the field towards a more rigorous use of these measurements. A thorough discussion of this literature has now been added to the Introduction and Discussion.

“Our observation of poor reliability questions the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid trait markers of the physiology of the oxytocin system in humans. […] Hence, our estimates of reliability are a better starter point for all studies where specific circumstances potentially affecting the activity of the oxytocin system have not been specified a priori.”

6) The paper misses a discussion of previous studies addressing links between salivary/ plasma levels and central oxytocin (e.g. in cerebrospinal fluid). I understand the claim that salivary oxytocin cannot be used to form an estimate of systemic absorption, although technically, a lack of a link between salivary and plasma levels, does not necessarily imply a lack of a relationship to e.g. central levels. The lack of effect is limited to this specific relationship.

In this study, we did not intend to investigate whether salivary and plasmatic oxytocin are valid proxies for the activity of the oxytocin system in the brain. Our data does not address that question and a thorough discussion of these studies falls, in our opinion, out of the scope of the manuscript. Instead, we focused on whether measurements of oxytocin in saliva and plasma (by far the most commonly used biological fluids to measure oxytocin) are sufficiently stable to provide valid indicators of the physiology of the oxytocin system in humans. Additionally, we also investigated whether salivary oxytocin can index plasmatic oxytocin at baseline and after the administration of synthetic oxytocin using different routes of administration.

A previous meta-analysis of studies correlating peripheral and CSF measurements of oxytocin has shown that most likely peripheral and CSF measurements do not correlate at baseline; significant correlations could be found after intranasal administration of oxytocin or specific experimental manipulations, such as stress(37). We believe that currently we still do not have a clear answer about the extent to which these peripheral fluids can actually index oxytocin concentrations in the brain (even if associations with CSF are evident in specific instances). For instance, no study has ever shown that CSF oxytocin actually predicts the concentrations of oxytocin in the extracellular fluid of the brain. Given what we currently know about the synaptic release of oxytocin in the brain(38) (in contrast with former theories of exclusive bulk diffusion in the CSF(39)), we think we have good reasons to suspect this might not be the case.

The only contribution our study can make in that respect is highlighting our current lack of understanding of how oxytocin reaches saliva if not from the blood. Currently there is no evidence of direct secretion of oxytocin to the saliva (not from acinar secretion or nerve terminals release). Hence, as it stands, the most likely mechanism for oxytocin to entry the saliva is from the blood (for instance, by ultrafiltration). If increases in plasmatic oxytocin after intravenous oxytocin cannot produce any significant increases in salivary oxytocin (shown in ours and in a previous study), how does oxytocin reach the saliva and why might it be able to predict concentrations in the CSF, if it does? In this respect, we hope our study highlights the need for further research shedding light on the mechanisms underlying these potential saliva – CSF relationships, if they exist. We would be glad to accommodate any other hypothesis the reviewer might have on this respect.

“The lack of increase in salivary oxytocin after the intravenous administration of exogenous oxytocin that was consistently found in our study and in a previous study(3) also raises the question of how oxytocin reaches the saliva if not from the blood. Currently there is no evidence of direct acinar secretion or direct nerve terminals release of oxytocin to the saliva; therefore, transport from the blood remains as the most plausible mechanism of appearance of oxytocin in the saliva. Clarifying these mechanisms of transport is paramount, given the current hypothesis that salivary oxytocin might be superior to plasma in indexing central levels of oxytocin in the CSF(40).”

Materials and methods

7) Related to the general comment, the variability in days between sessions is relatively high (average 8.80 days apart (SD 5.72; range 3-28). However, it appears that no explicit measures were taken to control the conducted analyses for this variability.

Thanks for point this out. Indeed, we were not sufficiently thorough in exploring the impact of this potential variability in the time gap between visits on our estimated ICCs. Thanks to the reviewer we now acknowledged this limitation of our analysis and decided to explore this further. We decided to run the following sensitivity analysis. First, we went back to our dataset A and identified all pairs of consecutive measures that were collected with an exact time interval of 7 days between visits. We could retrieve 15 examples of these pairs from 15 different participants for both saliva and plasma. Then, we recalculated the ICC and CV on this subset of our initial sample. In line with our main analysis, we found poor reliabilities for both salivary and plasmatic oxytocin; in both cases the ICCs were not significantly different from 0 and the CVs were 49% and 40%, respectively. This further analysis has been added to the revised version of the manuscript. We hope the reviewer shares our vision that our main conclusion of poor reliabilities of single measurements of baseline oxytocin in saliva and plasma cannot be simply attributed to the variability in the number of days between visits.

“Since there was considerable variability in the time-interval between visits across participants, we conducted a sensitivity analysis where we repeated our reliability analysis focusing on 15 pairs of consecutive measures that were collected with an exact time interval of 7 days between visits in 15 participants. Here, we recalculated the ICC and CV on this subset of our initial sample, using the approach described above.”

“These poor reliabilities are unlikely to be explained by variability in the time-interval between visits of the same individual, since we also found poor reliability indexes for both saliva and plasma when we restricted our analysis to a subset of our sample controlling for the exact number of days spacing visits.”

8) A rationale for the adopted dosing and timing (115 min post administration) of the sample extraction is missing. Additionally, it seems that intravenous administrations were always given second, whereas intranasal administrations were given third, with a small delay of approximately 5 min. Hence, it seems that the timing of 115 min post-administration is only accurate for the intranasal administration.

We collected saliva samples before any treatment administration and after the end of our scanning session (collection of saliva samples in between was just not possible because the participants were inside the MRI machine and could not have moved their heads). For the plasma, we collected samples before any treatment administration, after each treatment administration and at other five time-points during the scanning session. Here, we only report the plasma data that was acquired concomitantly with the saliva samples (the full-time course of plasma changes in plasmatic oxytocin has been reported elsewhere(2)).

In the manuscript, we report post-administration times from the end of the full treatment administration protocol. Hence, as the reviewer highlights our post-administration sample was collected at around 115 mins from the last intranasal administration and 120 mins from the end of the intravenous administration. We have now made this aspect explicit in the revised version of the manuscript.

“For the purposes of this report, we use the plasmatic and salivary oxytocin measurements that were obtained at baseline and at 115 minutes after the end of our last treatment administration (this means that our post-administration samples were collected 115 mins after the intranasal administrations and 120 mins after the intravenous administration of oxytocin).”

9) Since the ICC of baseline samples showed poor reliability, it seems suboptimal to pool across sessions for assessing the relationship between salivary and blood measurements. It should be possible to perform e.g. partial correlations on the actual scores, thereby correcting for the repeated measure (subject ID). Further, since the sample size is relatively small (13 subjects), it might be recommended to use non-parametric (e.g. Spearmann correlations) instead of Pearson. The additional reporting of the Bayes factor is appreciated; it is very informative.

Thanks for the suggestion. In fact, for the correlation the reviewer mentions we indeed used a multilevel approach where we specified subject as a random effect. This allowed us to deal with the dependence of measurements coming from the same subject in different visits. Furthermore, since we also had concerns about the sample size, we calculated Pearson correlations but used bootstrapping (1000 samples) to obtain the 95% confidence intervals and assess significance. Bootstrapping is a robust statistical technique which allows significance testing independently of any assumptions about the distribution of the data and is robust to outliers. Please see subsection “Association between salivary and plasmatic oxytocin levels”.

10) Now, the authors only compared relationships between salivary and plasma levels, either at baseline or post administration. I'm wondering whether it would be interesting to explore relationships between pre-to-post change scores in salivary versus plasma measures.

Thanks for the suggestion. We have now conducted this further analysis and we could not find any significant correlation between changes from baseline to post-administration in any of our treatment conditions. As for our other correlation analyses, here we also conducted Bayesian inference, which supported the idea that the null hypothesis of no significant correlation between changes in saliva and plasma from baseline to post-administration is at least 4x more likely than the alternative hypothesis. This further analysis strengthens our confidence that changes in salivary oxytocin after administration of oxytocin using the intranasal and intravenous routes should not be used to predict systemic absorption to the plasma.

“As a final sanity check, we also investigated correlations between the changes from baseline to post-administration in saliva and plasma in each of our treatment conditions separately.”

“Furthermore, we could not find any significant correlation between changes in salivary or plasmatic oxytocin from baseline to 115 mins after the end of our last treatment administration in any of our four treatment conditions. The lack of significant associations between salivary and plasmatic oxytocin (and respective changes from baseline) was further supported through our Bayesian analyses which demonstrated that given our data the null hypotheses were at least three times more likely than the alternative hypothesis.”

11) Please provide more information on the outlier detection procedure (outlier labelling rule).

This information has now been added to the revised version of the manuscript.

“Outliers were identified using the outlier labelling rule(41); this means that a data point was identified as an outlier if it was more than 1.5 x interquartile range above the third quartile or below the first quartile.”

12) Please indicate how deviations from a Gaussian distribution were assessed.

We used the combined assessment of i) differences between mean and median; ii) skewness and kurtosis; iii) histogram; iv) Q-Q plots; and v) the Kolmogorov-Smirnov and Shapiro-Wilk normality tests. Deviations from a normal distribution is common in the concentration of several analytes in the saliva (42), including oxytocin (15); hence, following the current recommendations, we used log transformations of the raw concentrations but plot the raw concentrations to facilitate the interpretation of our plots.

Results

13) Please verify the degrees of freedom for the post-hoc tests performed to assess pre-post changes at each treatment level (e.g. baseline vs Post administration: Spray – t(122) = 7.06, p < 0.001). Why is this 122? Shouldn't this be a simple paired-sample t-test with 13 subjects?

We apologize for this oversight. Indeed, we did a mistake in copying the values of the degrees of freedom from SPSS. We have now corrected these values. All the other p-values and F or T values were reported correctly and hence are not changed in the revised version of the manuscript (please see also response to reviewer 1, question 4 regarding inconsistencies in the reported p-values).

Reviewer #3:

• I would omit references to “biomarkers” or “biomarkers of the physiology of the oxytocin system” (e.g. in the title). I'm not sure which previous studies have claimed this link.

We have revised the text accordingly throughout the manuscript. Some examples below.

Title:

“Are single peripheral measurements of baseline oxytocin in saliva and plasma reliable trait markers of the physiology of the oxytocin system in humans?”

Abstract:

“However, questions remain about whether they are sufficiently stable to provide valid trait markers of the physiology of the oxytocin system, and whether salivary oxytocin can accurately index its plasmatic concentrations.”

“Conclusions: Our findings question the use of single measurements of baseline oxytocin concentrations in saliva and plasma as valid trait markers of the physiology of the oxytocin system in humans and suggest that, at best, these measurements can provide reliable state markers.”

Introduction

“Here, we investigate the second assumption, which is a prerequisite if single measurements of baseline levels of endogenous oxytocin are to be used as a valid trait markers of the physiology of the human oxytocin system(43).”

• In the last paragraph of the Introduction, only reference is made to assessing the effect of intravenous administration, whereas the study addresses both intravenous and intranasal effects.

Thanks for pointing that out. Indeed, we did not make full justice to the fact that our study investigated two different routes of administration (intranasal vs intravenous) and two intranasal methods (spray versus nebuliser). This information has been added to the Introduction in the revised version of the manuscript.

“Here, we aimed to characterize the reliability of both salivary and plasmatic single measures of basal oxytocin in two independent datasets, to gain insight about their stability in typical laboratory conditions and their validity as trait markers for the physiology of the oxytocin system in humans. Additionally, we investigated whether salivary oxytocin concentration reflects plasmatic oxytocin by examining i) if the intravenous administration of exogenous oxytocin increases the concentration of salivary oxytocin; ii) how potential changes in salivary oxytocin compare between different routes of administration (intranasal versus intravenous) and methods of intranasal administration (spray versus a nebuliser); and iii) the correlation between plasmatic and salivary oxytocin levels at baseline and after the administration of exogenous oxytocin using two different methods of intranasal administration (spray versus nebuliser) and the intravenous route.”

• In the Materials and methods it is stated that “participants had no history of psychiatric disorders or substance abuse”. How was this assessed? Did the authors adopt any particular questionnaire or scale?

We apologized for not having described this in sufficient detail. In dataset A, we screened participants for psychiatric conditions using the Symptom Checklist-90-Revised(44) and the Beck Depression Inventory-II(45) questionnaires. In dataset B, participants were screened using the MINI International Neuropsychiatric Interview(46). This information has been added to the revised version of the manuscript.

“All participants had no history of psychiatric disorders or substance abuse, scored negatively on a screening test for recreational drug use, and did not currently use any medication. In dataset A, we screened participants for psychiatric conditions using the Symptom Checklist-90-Revised(44) and the Beck Depression Inventory-II(45) questionnaires. In dataset B, we used the MINI International Neuropsychiatric Interview(46).”

• Please indicate explicitly the number of subjects involved in any of the (Pearson) correlation analyses e.g. assessing relationships between salivary and plasma measurements.

The exact number of subjects used for each correlation analysis has now been added to the revised version of the manuscript.

• In the Discussion, it is indicated that “the time interval between measurements do not seem to significantly impact on the reliability of baseline oxytocin”. This was not explicitly assessed.

Indeed, we did not explain in detail how we evaluated this aspect. This conclusion derives from the comparison of the overlap of the 95% confidence intervals for the ICCs estimated for each pair of sessions. As the reviewer can see in Supplementary file 2, there is considerable overlap between the 95% CI of the different ICCs. This suggests that there are no significant differences between ICCs across different pairs of visits, even if we find numerically higher ICCs for the following plasma pairs visits 1-2 and visits 3-4. We now explain this in further detail in the revised version of our Discussion.

“The time-interval between measurements do not seem to significantly impact on the reliability of baseline oxytocin, as suggested by the overlap of the 95% confidence intervals of the ICCs estimated for each pair of sessions.”

• Abbreviations in figures need to be reported in full in the figure legend.

Thanks for pointing that out. We have revised the legend of Figure 1 to report the abbreviations missing.

• Figure 2. It would be helpful to use the same range for the y-axis of the plasma assessments (in sample A and B).

We have implemented the suggestion in the revised version of Figure 2.

• It would be recommended to use a consistent order for reporting the different treatment levels. Now the table in Figure 1 uses the order spray, IV, placebo, Nebulizer, whereas in Figure 2, this order is changed to IV, nebulizer, placebo, spray. I guess the most logic order would be spray, nebulizer, IV and placebo (as used in-text).

Thanks for pointing out this inconsistency. In the revised version of our figures and tables we now use a consistent order, which matches the order we used in-text.

• It is unclear why the presentation modes for presenting the associations between salivary and plasma OT at baseline (Figure 4) and post-administration (Figure 5) are different. It would be informative to plot for both the regression lines and distribution plots.

We decided to not include the marginal distribution plots in Figure 5 to make the figure easier to read. We have now revised Figure 5 to match the mode of presentation of Figure 4.

• Supplementary figures need figure legends.

These legends were presented at the end of the manuscript as recommended by eLife. eLife does not accept the inclusion of text in supplementary.

• I would recommend omitting the reporting of “dataset B” from the main manuscript, or only report it (briefly) as a secondary analysis, with some additional information in the supplements.

Thanks for the suggestion, but as explained in the point just above, eLife does not accept the inclusion of text in supplementary.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Reviewer #1:

This is a revision of an article that I previously reviewed investigating if single peripheral measurements of baseline oxytocin in saliva and plasma are reliable trait markers of the physiology of the oxytocin system in humans.

The paper has now improved and most of my original queries have now been satisfactorily addressed.

However, I still have one comment regarding the author's response to query #2 "It is important to note that the 1IU intravenous dose in this study led to equivalent concentrations in blood compared to intranasal administration": I now better understand the justification for using a 10IU dose (i.e., "we demonstrate that even when plasmatic levels of OT are maintained substantially increased throughout the observation interval, we cannot detect increases in salivary oxytocin". However, this should also be better emphasized in the manuscript.

Thank you for your previous helpful comments, we are glad we were able to address your comments and that you find that the manuscript has improved. Thank you also for the opportunity to clarify this remaining point. We have now highlighted this aspect of our design in the Introduction and Materials and methods sections of the revised version of our manuscript (see below).

“Additionally, we investigated whether salivary oxytocin concentration reflects plasmatic oxytocin by examining i) if the intravenous administration of a large dose of oxytocin which produces sustained increases in plasmatic oxytocin over the course of two hours also increases the concentration of salivary oxytocin;”<bold />

“The administration of 10IU of oxytocin intravenously produces sustained increases in the levels of plasmatic oxytocin over a two hours course(1). This aspect of our design allows us to eliminate the possibility that the lack of changes in salivary oxytocin is due to under-dosing.”

Reviewer #3:

Overall, the authors were able to provide additional information and conduct additional analyses that provided solutions to several of the raised methodological concerns (e.g. regarding timing of saliva collection, within-session reliability, sample size/ power, and other statistical remarks). Some other methodological issues may still need further clarification however, such as the possible impact of variability in collected sample volume across sessions (at least for the salivary samples). Also, considering the main research question of the current study (reliability of oxytocin sampling), it would be recommended that a measure of intra-assay variability was available for the own sample collections. The efforts for accounting for the effect of variability in between-session intervals are appreciated. I'm wondering however, whether it would be possible to perform secondary analyses on the whole data set (rather than performing subset analyses) regressing out possible effects of variability in between-session intervals.

In general, I feel that the manuscript already improved significantly compared to the initial submission, increasing its potential for publication tremendously. However, some of the raised concerns regarding the relative novelty regarding the study design and conclusions may still remain, raising questions whether a more specialized journal may be more suitable for its publication.

Thank you for your previous helpful suggestions and comments. We are glad that the additional information and analyses addressed most of the methodological concerns and that you consider that the manuscript has substantially improved. Thank you also for the opportunity to clarify any remaining concerns, which we address below.

As a further clarification of the novelty of our study, we would like to highlight that our study is the first of its kind to evaluate the question of the validity of single peripheral measurements of baseline oxytocin in saliva and plasma as reliable trait markers of the physiology of the oxytocin system in humans using the appropriate methods for the quantification of oxytocin concentrations and the appropriate statistical approach to assessing reliability. Thanks to our study, we came to appreciate that most likely previous claims that single measurements of baseline oxytocin in saliva and plasma are sufficiently stable within individuals to provide a valid trait marker of the physiology of the oxytocin system were probably misguided. Our second question related to the validity of salivary oxytocin to index oxytocin concentrations in the plasma is indeed an extension of previous work; here, critical aspects of our design were novel and allowed us to expand previous findings in important ways (namely, the use of a high dose of intravenous oxytocin which eliminated the possibility that the lack of an association might have been driven by under-dosing, and a method for oxytocin quantification that was sufficiently sensitive to the baseline physiological range of oxytocin).

Regarding the specific points raised:

1) Possible impact of variability in collected sample volume across sessions (at least for the salivary samples).

We have now provided further details in the manuscript that clarify the standard operating procedures for blood sample and saliva collection. In both cases we followed the standard operating procedures as provided by Professor Rainer Landgraf (RIAgnosis, standard operating procedures available upon request).

Blood samples: The collected volume of blood was the same across participants and sessions since we used standard 5ml EDTA vacutainer tubes. Following centrifuging, exactly 0.5 ml plasma was aliquoted in 2ml Eppendorf vials (and stored until analysis as described in the manuscript).

Saliva: We collected saliva using Salivettes (Sarstedt 51.1534.500), which were then centrifuged and exactly 0.5ml aliquots of saliva was pipetted to 1.5ml Eppendorf vials and stored until analysis. This was the same across participants and sessions. As expected when collecting saliva with Salivettes, small variations in the initial volume of saliva collected before centrifugation may have existed. However, we do not think this could induce considerable artificial variability in the concentrations of salivary oxytocin between sessions. First, we measured concentrations of oxytocin (pg of oxytocin per millilitre) in the exact same centrifuged volume for all participants and sessions. The salivette allows for a recovery of mean saliva volumes in the range of 1.1 ± 0.3 ml, which is at least 2 x higher than the minimal volume we would need to quantify oxytocin. Previous studies have only raised concerns about the recovery of the concentrations of small peptides in saliva samples collected with salivettes when the initial collected volumes are lower than 0.25 ml(2, 3), which was not the case in our study. We have now included these clarifications in the revised version of the manuscript (see below).

“Blood was collected in 5ml ethylenediaminetetraacetic acid vacutainers (Kabe EDTA tubes 078001), placed in iced water and centrifuged at 1300 × g for 10 minutes at 4°C within 20 minutes of collection and then 0.5ml of plasma was immediately pipetted into 2ml Eppendorf vials. […] Minimizing the time-interval samples were kept in the collection devices also allowed us to keep potential absorption to the walls of these recipients to a minimum(2).”

2) Obtaining a measure of intra-assay variability for own sample collections: We agree with the reviewer, and thanks to their previous suggestion we estimated variability in our own sample using two sample acquisitions that were obtained within 15 mins from each other in the placebo visit. Our within-session reliability analysis showed excellent reliability (ICC=0.92, CV=20%). We hope that the reviewer agrees that this is reassuring that the poor reliability across sessions we report here is unlikely to have resulted simply from measurement error.

3) The efforts for accounting for the effect of variability in between-session intervals are appreciated. I'm wondering however, whether it would be possible to perform secondary analyses on the whole data set (rather than performing subset analyses) regressing out possible effects of variability in between-session intervals.

Thank you for this suggestion and your appreciation of our effort to account for the effect of variability in between-session intervals. We understand that the reviewer wonders to what extent variability in the within-subject intervals between samples might contribute to the lack of reliability that we report. Following discussions with colleagues and a statistician, we addressed this questions through three possible lines of analyses, which all provided converging results:

a) The previously reported sensitivity analyses. The sensitivity analysis we presented in the last version of the manuscript, where we examined the reliabilities for both salivary and plasmatic oxytocin in a subset of our sample where two consecutive saliva and plasma samples were collected with an exact gap of seven days. For both plasma and saliva, the estimated ICCs were not significantly different from 0 and the CVs were 40% and 49%, respectively.

b) The previously reported pairwise comparisons (e.g. visit 1 vs visits 2/3/4), where the interval between sample acquisition may not have been exactly fixed, but it systematically increased with each visit. As we reported, we did not find any significant effect of time-interval on our estimated ICCs. If time-interval was driving the poor reliabilities, then we would have expected that in our pairwise analyses reliability would be consistently higher for samples closer in time and drop as the time-interval between sessions increases. This was not what we found (please see Between-visits reliability analysis for each pair of visits in our manuscript).

c) Additionally, we conducted a new analysis using information from the whole sample, as the reviewer suggested. Here, we explored the potential impact of variability in the time interval between sessions as follows. The ICC is the ratio of between-participant variance to total variance, which in turn is the sum of within- and between-participant variance. Therefore, one possible way to address the contribution of variability in the within-subject intervals between samples is to examine the relationship between within-participant variance in oxytocin concentrations and within-participant variance in the time interval between sample acquisitions, across participants. We did this and found that correlations between oxytocin and time interval variances were non-significant for both plasma (Spearman Rho = 0.406, p = 0.118) and salivary measurements (Spearman Rho = -0.524, p = 0.065). (For salivary measurements, it may be worth noting the negative sign of the correlations, suggesting that increased variability in time interval would predict, if significant, decreased variability in oxytocin measurements, which is counterintuitive. We know better of course than interpreting non-significant results, especially when inconsistent in direction which suggests random estimates around a mean of 0 association between variances).

We believe that these three lines of analysis provide converging evidence that variability in the time intervals between acquisitions is unlikely to be driving the poor ICCs we report in this manuscript (see below for the changes in the revised version of the Discussion). This converging evidence is consistent with our main conclusion that single peripheral measurements of oxytocin at baseline may not provide valid trait markers of the physiology of the oxytocin system and we that hope the reviewer agrees that the variability in time-interval (at least within the range of days examined in this study) between sessions is unlikely to be a key driver of the low reliabilities we report here.

Variance in the within-subject intervals between samples did not correlate with within-participant variance in oxytocin concentrations across participants neither for plasma (Spearman Rho = 0.406, p = 0.118) or saliva (Spearman Rho = -0.524, p = 0.065).”

These poor reliabilities are unlikely to be explained by variability in the time-interval between visits of the same individual. Three lines of converging evidence support this conclusion. First, we also found poor reliability indexes for both saliva and plasma when we restricted our analysis to a subset of our sample controlling for the exact number of days spacing visits. Second, we did not find any significant effect of time-interval on our estimated ICCs. If time-interval was driving the poor reliabilities, then we would have expected that in our pairwise analyses reliability would be consistently higher for samples closer in time and drop as the time-interval between sessions increases. This was not what we found. Third, variability in the within-subject intervals between samples did not correlate with within-participant variance in oxytocin concentrations across participants.”

Since there was considerable variability in the time-interval between visits across participants, we conducted a sensitivity analysis where we repeated our reliability analysis focusing on 15 pairs of consecutive measures that were collected with an exact time interval of 7 days between visits in 15 participants. Here, we recalculated the ICC and CV on this subset of our initial sample, using the approach described above. We also investigated whether within-participant variance in the time interval between sample acquisitions could predict within-participant variance in oxytocin concentrations across participants, using Spearman correlations.”

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https://doi.org/10.7554/eLife.62456.sa2

Article and author information

Author details

  1. Daniel Martins

    Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0239-8206
  2. Anthony S Gabay

    1. Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom
    2. Centre for Human Brain Health, University of Birmingham, Birmingham, United Kingdom
    Contribution
    Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6946-1046
  3. Mitul Mehta

    1. Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom
    2. Centre for Human Brain Health, University of Birmingham, Birmingham, United Kingdom
    Contribution
    Supervision, Funding acquisition, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Yannis Paloyelis

    Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, Kings College London, London, United Kingdom
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing
    For correspondence
    yannis.paloyelis@kcl.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4029-3720

Funding

Economic and Social Research Council (ES/K009400/1)

  • Yannis Paloyelis

NIHR Biomedical Research Centre, South London and Maudsley

  • Mitul Mehta
  • Yannis Paloyelis

PARI GmbH

  • Yannis Paloyelis

IoPPN, King’s College London

  • Anthony S Gabay

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Sofia Vasilakopoulou and Jack Loveridge for their assistance in data collection. We also thank Rosa Oliveira and Silia Vitoratou for their advice on statistical analysis. Most importantly, we thank all participants volunteering to both studies. This study was part-funded by: an Economic and Social Research Council Grant (ES/K009400/1) to YP; scanning time support by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London to YP; an unrestricted research grant by PARI GmbH to YP. Data collection for dataset B was supported by an IoPPN-MRC Excellence Studentship awarded to AG. MAM is in part supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. Disclosures YP, DM, MM, and AG declare no competing financial interests. MM received research funding from Takeda and Lundbeck and support in kind from Johnson and Johnson and AstraZeneca. This manuscript represents independent research. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health and Social Care, or PARI GmbH.

Ethics

Human subjects: All participants gave informed consent prior to testing. King's College London Research Ethics Committee approved the protocols for both studies (Dataset A: PNM/13/14-163; Dataset B: PNM/14/15-32).

Senior Editor

  1. Christian Büchel, University Medical Center Hamburg-Eppendorf, Germany

Reviewing Editor

  1. Joseph G Gleeson, Howard Hughes Medical Institute, The Rockefeller University, United States

Publication history

  1. Received: August 25, 2020
  2. Accepted: December 3, 2020
  3. Version of Record published: December 11, 2020 (version 1)

Copyright

© 2020, Martins et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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